Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1015202530CA 02264450 1999-03-05MSH File No. GFP CA_'l_â[I_âI£: Methods for Monitoring Heterologous Sex ChromosomesFIELD OF THE INVENTIONThe invention relates to methods for monitoring heterologous sex chromosomes. Theinvention also relates to pluripotent cells comprising a nucleic acid sequence encoding aï¬uorescent protein marker selectively integrated into a heterologous sex chromosome in thecell; embryos and transgenic animals produced using the pluripotent cells; and, the uses ofsuch cells, embryos, and animals, in particular in monitoring heterologous sexchromosomes.BACKGROUND OF THE INVENTIONIn many species the male and female sex is determined by the segregation ofspecialized chromosomes i.e. sex chromosomes. There has been a long time interest tocontrol this random process in many species of wild, farm, and laboratory animals. Thereasons for controlling sexing include reducing the incidence of sexâlinked genetic disorders,and more efficient animal production. For example, the bovine dairy and beef agriculturesectors need female and male animals respectively, and the undesired animal is culled.Selection of animals of a desired sex has been carried out by sexing ofpreimplantation embryos, or separation of X and Y bearing spermatozoa. Preimplantationembryo sex selection has been accomplished using karyotyping, amplification of Y-chromosome specific nucleotide sequences, or immunological methods. The first twomethods are invasive methods since they involve embryo micromanipulation.Immunological methods are typically based on immunodetection of a male-specificmarker. Eichwald and Silmser (1955, Transplant Bull 2: 148) found that within the inbredmouse strain C57BL/6, skin transplants from males to females were rejected, whereastransplants from males to males, females to males and females to females within the samestrain were tolerated. These results were attributed to an antigen coded for by a Yâlinkedgene, and the system came to be known as HâY (histocompatibility locus on the Ychromosome) (Hauscha (1955, Transplant Bull 2:154). Antibodies specific for HâY are usedin sexing studies e.g. enzymeâlinked immunoabsorbent assay (ELISA) methods [Bradley etal. (1987, Hum. Genet. 762352) and Brunner and Wachtel (1988, J. Irnmunol. Methods106:49). See also Epstein, (1980, Tiss. Antigens 15:63); Anderson, (1987, Theriogenology27:81); Wachtel, (1988,Fe1t. Ster. 50:355); Avery and Schmidt, (1989, Acta. Vet. Scand.302155) for the use of HâY antibodies to sex embryos]. However, it is uncertain that HâY is1015202530CA 02264450 1999-03-05-2-preferentially expressed on Y-bearing sperm (e.g. Hendricksen et al. 1993, Mol. Reprod.Devel. 352189) and, some investigators have concluded that differences between the twoclasses of sperm can not be detected immunologically (Windsor et al. (1993, Reprod. Fert.Dev. 5:155).Further, a âfemale protein" which is hormoneâdependent and therefore unlikely tobe found in blastocysts or sperm has been reported by Brown et al. (1991, Nature 349: 38).An XX-specific molecule which is an mRNA molecule transcribed by the "inactivated" X,and therefore only produced in females in somatic tissues has also been reported by Coe(1977, Proc. Nat. Acad. Sci. 74: 730).Cell markers have been used in transgenic animal production to select embryos thathave an integrated transgene. Typical selection methods employ reporter genes such asluciferase, Bâgalactosidase, and alkaline phosphatase that are not particularly useful sincethey require harmful substrates for their detection. Other methods involve PCR analysis ofblastomere biopsies which requires time and complicated micromanipulation. There is alsoa high risk that an embryo will be damaged using these selection methods.Non-invasive methods have been reported for selection of transgeneâintegratedembryos using green fluorescent protein (GFP) as a marker (Takada, T., et al, NatureBiotechnology 152458, 1997). The method involves microinjecting embryos with a GFPtransgene, identifying GFPâpositive blastocysts, and transferring the blastocysts to uteri ofpseudopregnant females to produce transgenic mice. The method has a number ofdisadvantages. In some studies, it has not been possible to obtain fetuses from blastocyststhat strongly expressed GFP (Takada, T. et al, supra). Fetus production rates have beenreported to be less than 25% and have been postulated to be the result of the damage causedby microinjection and gene integration. Further, in a number of studies microinjection ofwildâtype GFP has not resulted in ubiquitous expression of GFP (H. Niwa et al Gene108:193-199, 1991; M. Ikawa, FEBS Lett 375:125â128, 1995; and M. Ikawa, et al, Dev.Growth Diff 37:455-459, 1995). Others have used mutant GFPs and have reported a visiblesignal in all tissues with the exception of erythrocytes and hair (M. Okabe et al, FEBSLetters 407:313â319, 1997). One mutant form of GFP (MmGFP) has been used to make anembryonic stem cell line expressing MmGFP, and the cells have been introduced into mouseembryos (ZernickaâGoetz M., et al Development 124:1133-1137, 1997).Cell markers that require substrates have been used to monitor X chromosomeactivity at the single cell level. In particular, an Xâlinked lacZ transgene has been used to1015202530CA 02264450 1999-03-05-3-examine the progression of Xâinactivation in different somatic lineages (Tam, P. et alDevelopment 12012925-2932, 1994).SUMMARY OF THE INVENTIONBroadly stated the present invention relates to a pluripotent cell comprising a nucleicacid sequence encoding a ï¬uroescent protein marker selectively integrated into aheterologous sex chromosome in the cell. The invention also provides a pluripotent cell linecomprising pluripotent cells of the invention. In particular an embryonic stem cell line isprovided comprising embryonic stem cells of the invention. Further, the invention providesa chimeric embryo comprising pluripotent cells of the invention, in particular embryonicstem cells of the invention.The invention also provides a method of producing a pluripotent cell that has aheterologous sex chromosome linked ï¬uorescent protein marker comprising introducing intoa pluripotent cell a nucleic acid sequence encoding a ï¬uorescent protein marker, andoptionally a nucleic sequence encoding a protein of interest, under conditions so that theï¬uorescent protein marker is selectively integrated into a heterologous sex chromosome inthe cell.In an embodiment of the invention a method is provided for producing anembryonic stem cell that has an Xâlinked ï¬uorescent protein marker comprising introducinginto embryonic stem cells a nucleic acid sequence encoding a ï¬uorescent protein marker,and optionally a nucleic sequence encoding a protein of interest, under conditions so that theï¬uorescent protein marker is selectively integrated into the X chromosome in the cell.The invention also contemplates a method of producing a chimeric embryo thatdevelops into a nonâhuman animal that transmits to its progeny a nucleic acid sequenceencoding a ï¬uorescent protein marker selectively integrated into one of the heterologous sexchromosomes in the animalâs cells comprising:(a) providing pluripotent cells, in particular embryonic stem cells, comprising a nucleicacid sequence encoding a ï¬uorescent protein marker selectively integrated into oneof the heterologous sex chromosomes in the cell; and(b) introducing the pluripotent cells in an embryo or aggregating the pluripotent cellswith an embryo to produce a chimeric embryo.A chimeric embryo may be implanted into a pseudopregnant foster mother, and allowedto grow to term to produce a nonâhuman transgenic animal that is capable of transmitting1015202530CA 02264450 1999-03-05-4-the nucleic acid sequence only to female or male progeny. Therefore, the invention alsocontemplates a non-human transgenic animal having a ï¬uorescent protein marker selectivelyintegrated into one of the heterologous sex chromosomes in its cells.The invention still further provides a method for producing a non-human animalhaving a ï¬uorescent protein marker integrated into one of the heterologous sexchromosomes in its cells comprising;(a) selecting an embryo that contains cells with a nucleic acid sequence encoding aï¬uorescent protein marker selectively integrated into one of the heterologous sexchromosomes in the cells;(b) allowing the embryo to develop to term to produce a non-human animal; and(c) optionally breeding the non-human animal and selecting progeny that express theï¬uorescent protein marker.In an embodiment of the invention a method is contemplated for producing a non-human animal, preferably a rodent, having a ï¬uorescent protein marker selectively integratedinto one of the sex chromosomes in its cells comprising the steps of:(a) introducing into an embryo of the animal, pluripotent stem cells comprising a nucleicacid sequence encoding a ï¬uorescent protein marker selectively integrated into one ofthe heterologous sex chromosomes in the cells;(b) allowing the embryo to develop to term to produce a non-human animal having aï¬uorescent protein marker selectively integrated into one of the heterologous sexchromosomes in its cells; and(c) optionally breeding the animal wherein one of the male or female progeny express theï¬uorescent protein marker.The non-human animals of the invention may be bred to normal animals or otheranimals carrying a second transgene(s) to create an animal carrying two transgenes i.e. adouble transgenic animal.The invention also provides a method of monitoring expression of a protein ofinterest linked to one of the heterologous sex chromosomes in a non-human animalcomprising:(a) introducing into pluripotent cells a transgene comprising a DNA sequenceencoding a protein of interest and a sequence encoding a ï¬uorescent protein1015202530CA 02264450 1999-03-05-5-marker under conditions so that the ï¬uorescent protein marker is selectivelyintegrated into a heterologous sex chromosome in the cells;(b) culturing the pluripotent cells in conditions permitting expression of theï¬uorescent marker protein and the gene of interest; wherein the pluripotent cellsexpressing the fluorescent marker protein express the gene of interest; and(c) monitoring the expression of the gene by monitoring expression of theï¬uorescent protein marker in the pluripotent cells.The pluripotent cells (e. g. embryonic stem cells), may be injected into a blastocystor aggregated with an early stage embryo to produce a chimeric embryo and the expressionof the protein may be monitored by monitoring expression of the ï¬uorescent protein markerin the chimera or in the growing chimera. The chimera may be implanted into apseudopregnant foster animal and the expression of the gene may be monitored bymonitoring expression of the ï¬uorescent protein marker in the growing chimera or in theresultant transgenic animal.The invention also provides a method of selecting embryonic stem cells containinga preselected heterologous sex chromosome comprising(a) introducing into embryonic stem cells a transgene comprising a sequenceencoding a ï¬uorescent protein marker under conditions so that the ï¬uorescentprotein marker is selectively integrated into the heterologous sex chromosomesin the cells;(b) culturing the embryonic stem cells in conditions permitting expression of theï¬uorescent protein marker; and(c) selecting embryonic stem cells containing the heterologous sex chromosome bydetecting embryonic stem cells expressing the ï¬uorescent protein marker.The invention further provides a method of selecting male or female embryoscomprising: producing embryos containing a ï¬uorescent protein marker selectivelyintegrated into a preselected heterologous sex chromosome; and selecting male or femaleembryos depending on whether the embryos express the ï¬uorescent protein marker.Other objects, features and advantages of the present invention will become apparentfrom the following detailed description. It should be understood, however, that the detaileddescription and the specific examples while indicating preferred embodiments of the1015202530CA 02264450 1999-03-05-6-invention are given by way of illustration only, since various changes and modificationswithin the spirit and scope of the invention will become apparent to those skilled in the artfrom this detailed description.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1A is a photograph showing the expression of EGFP in ES cells;Figure 1B is a photograph showing the expression of EGFP in drug-resistantcolonies, exhibiting ubiquitous EGFP expression;Figure 1C is a photograph showing the expression of EGFP in differentiated EScells;Figure 1D is a photograph showing embryoid bodies exhibiting ubiquitoustransgene activity;Figure 2A is a photograph showing ES cells colonizing part of, and the majorityof morula stage embryos;Figure 2B is a photograph showing ES cells colonizing most of the ICM but notthe trophectoderm of a blastocyst stage embryo;Figure 2C is a photograph of ES cells colonizing a proportion of the inner cellmass (ICM) of a blastocyst;Figure 2D is a photograph of ES cells colonizing a proportion of the inner cellmass of a blastocyst;Figure 2E is a photograph of ES cells localized to the majority of the ICM with asingle cell in the trophectoderm of a blastocyst;Figure 2F is a photograph of ES cells localized to part of the ICM with two cellsseen in the trophectoderm;Figure 3A is a schematic diagram of a method detailing the technique of diploidgreen ES cell wild type embryo aggregations;Figure 3B is a photograph showing the aggregation of a clump of greenfluorescent ES cells with a wild-type embryo to form a blastocyst after overnightincubation;Figure 3C is a photograph showing the aggregation of a clump of greenï¬uorescent ES cells with a wild-type embryo to form a blastocyst after overnightincubation;Figure 3D is a photograph showing the aggregation of a clump of green1015202530CA 02264450 1999-03-05-7-ï¬uorescent ES cells with a wild-type embryo to form a blastocyst after overnightincubation;Figure 3E is a photograph showing the aggregation of a clump of greenfluorescent ES cells with a wild-type embryo to form a blastocyst after overnightincubation, with preferential contribution of the ES cells to the inner cell mass;Figure 4A is a photograph of a postimplantation embryo showing the ventralview of a late headfold/presomite stage (E8) embryo;Figure 4B is a photograph of a postimplantation embryo showing a lateral view ofan early somite stage (E8.5) embryo;Figure 4C is a photograph of a postimplantation embryo showing a dorsal View ofan E8.5 embryo;Figure 4D is a photograph of a postimplantation embryo showing a lateral view ofthe head and forelimb of a new-bom chimera;Figure 5A is a photograph of oneâ to four-cell stage embryos exhibiting notransgene expression;Figure 5B is a photograph of embryos showing initiation of expression at themorula stage;Figure 5C is a photograph of E3.5 embryos in dark-field;Figure 5D is a photograph of E3.5 embryos in bright-field;Figure SE is a photograph of two-cell and morula stage greenâï¬uorescentembryos in bright-field;Figure 5F is a photograph of two-cell and morula stage greenâfluorescent embryosin dark-field;Figure 5G is a photograph of blastocyst stage embryos in dark-field;Figure 5H is a photograph comparing embryos derived from a hemizygous malecrossed to a wild-type female and a wild-type male crossed to a hemizygous female;Figure 6A is a photograph of an E4.75 newly hatched and implanted blastocystattached to decidual tissue;Figure 6B is a photograph showing B5/EGFP expression in an E5 embryo;Figure 6C is a photograph showing B5/EGFP expression in an E5.5 embryo;Figure 6D is a photograph showing B5/EGFP expression in an E9 embryo withassociated ectoplacental cone partially contained within the deciduum;Figure 6E is a photograph showing B5/EGFP expression in an E9 extraembryonic1015202530CA 02264450 1999-03-05membrane;Figure 6F is a photograph of two E9.5 litteimates in brightâfield;Figure 6G is a photograph in dark-field of the two embryos shown in Figure 6F;Figure 6H is a photograph showing B5/EGFP expression in an E11.5 embryowith placenta;Figure 61 is a photograph showing B5/EGFP expression in an E11.5 embryo heartand lungs;Figure 6] is a photograph showing B5/EGFP expression in an E11.5 embryokidney rudiment and intestine;Figure 7A is a photograph showing ubiquitous green ï¬uorescence in the head andforelimb of a newâbom B5/EGFP hemizygote pup among nonâtransgenic littermates;Figure 7B is a photograph showing ubiquitous green fluorescence in a newâbomB5/EGFP hemizygote pup and nonâtransgenic littermate;Figure 7C is a photograph showing ubiquitous green ï¬uorescence in 1-weekâoldtransgenic and nonâtransgenic pups;Figure 7D is a photograph showing ubiquitous green ï¬uorescence in an anteriorview of a 3âweekâold albino B5/EGFP mouse;Figure 7E is a photograph showing ubiquitous green ï¬uorescence in a posteriorview of a 3âweekâold albino B5/EGFP mouse;Figure 7F is a photograph of a posterior view of the tails of 2-month albino,chinchilla, agouti mice illustrating pigmentâdependent ï¬uorescence;Figure 7G is a photograph of a dorsal view of an incision made in a 3âweekâoldB5/EGFP mouse body with skin removed to reveal ï¬uorescence in the body;Figure 7H is a photograph of brains from 3âweekâold transgenic mouse and non-transgenic littermates;Figure 71 is a photograph of kidneys from 3âweekâold transgenic mouse and non-transgenic littermates;Figure 7] is a photograph of livers from 3âweekâold transgenic mouse and non-transgenic littermates;Figure 7K is a photograph of pancreas from 3âweekâold transgenic mouse andnonâtransgenic littermates;Figure 7L is a photograph of intestines from 3âweekâold transgenic mouse andnonâtransgenic littermates;1015202530CA 02264450 1999-03-05-9-Figure 8A is a schematic diagram detailing the tetraploid technique;Figure 8B is a photograph showing the progression of an aggregation betweengreen ES cellsâwildâtype embryo to the blastocyst stage;Figure 8C is a photograph showing the progression of an aggregation betweengreen ES cellsâwildâtype embryo to the blastocyst stage;Figure 8D is a photograph showing the progression of an aggregation betweengreen ES cellsâwildâtype embryo to the blastocyst stage;Figure 8E is a photograph showing the progression of an aggregation betweengreen ES cellsâwi1dâtype embryo to the blastocyst stage;Figure 9A is a photograph of a green ï¬uorescent E9 embryo with non-ï¬uorescentextraâembryonic membranes;Figure 9B is a photograph of a green ï¬uorescent E11 embryo with mosiac yolksac and non-ï¬uorescent placenta;Figure 9C is a photograph of an E8.5 embryo with green ï¬uorescentextraembryonic membranes;Figure 9D is a photograph of an E11 embryo with mosaic yolk sac andï¬uorescent placenta;Figure 10A is a photograph of new-bom pups from a cross between a B5/EGFPhemizygous male and a wild-type ICR female;Figure 10B is a photograph of mice from a litter of pups derived from anintercross between a male and female both of which were hemizygous for the transgene;Figure 10C is a photograph of a close view of pups hemizygous and homozygousfor the trans gene;Figure 10D is a photograph of the tail tips from E1 1.5 embryos homozygous,hemizygous, and nonâtransgenic;Figure 11A is a photograph showing a litter of F1 offspring derived from a crossbetween a D4/XEGFP transgenic male and a wild type female;Figure 11B is a photograph of 6 pups from a litter derived from a cross between ahemizygous female and wild-type male;Figure 12A is a photograph of an E6.5 embryo obtained from matings between aD4/XEFGP transgenic male and a wild-type ICR female, and a non-transgenic embryo;Figure 12B is a photograph of an E8.5 embryo obtained from matings between aD4/XEFGP transgenic male and a wild-type ICR female, and a non-transgenic embryo;1015202530CA 02264450 1999-03-05-10-Figure 12Cis a photograph of an E10.5 embryo obtained from matings between aD4/XEFGP transgenic male and a wildâtype ICR female, and a non-transgenic embryoFigure 13A is a photograph of a pool of three litters of E3.5 embryos derivedfrom natural matings between transgenic males and wild type females in bright field,where green ï¬uorescent embryos are female and the nonâfluorescent embryos are male;Figure 13B is a photograph of a pool of three litters of E3.5 embryos derivedfrom natural matings between transgenic males and wild type females in dark field withbackground illumination, where green fluorescent embryos are female and the non-fluorescent embryos are male;Figure 13C is a photograph of a pool of three litters of E3.5 embryos derivedfrom natural matings between transgenic males and wild type females in full dark field,where green ï¬uorescent embryos are female and the non-ï¬uorescent embryos are male;Figure 13D is a photograph of color and sex pooled embryos which gave rise tosingle sex litters after transfer to surrogate females;Figure 14A is a photograph in bright-field of litters of E3.5 embryos derived froma cross between a D4/XEFGP transgenic male and a wildâtype ICR female;Figure 14B is a photograph in dark field of litters of E3.5 embryos derived from across between a D4/XEFGP transgenic male and a wildâtype ICR female;Figure 14C is a photograph of litters of E10 embryos derived from a crossbetween a D4/XEFGP transgenic male and a wildâtype ICR female; andFigure 14D is a gel showing PCR genotyping for the presence of a Ychromosome using primers for Sry and a control assay for the presence of an autosomalgene using primers for the myogenin gene.DETAILED DESCRIPTION OF THE INVENTIONAs hereinbefore mentioned, the present invention relates to a pluripotent cellcomprising a nucleic acid sequence encoding a ï¬uroescent protein marker selectivelyintegrated into one of the heterologous sex chromosomes in the cell.The pluripotent cell may be any totoipotent, pluripotent embryonic/somatic cells,embryonic stem cells and totipotent somatic cell cultures, which are all gerrnline compatible.Primary isolates of embryonic stem cells may be used that are obtained directly fromembryos such as the CCE cell line (Robertson, E.J. In: Current Communications inMolecular Biology, Capecchi, M.R. (ed), Cold Spring Harbor Press, Cold Spring Harbor,1015202530CA 02264450 1999-03-05-11-N.Y. (1989), p39-44) or from clonal isolation of embryonic stem cells from such cell lines(Schwartzberg, P.A. et al., Science 246:799-803, 1989; E.J. Robertson In: Teratocarcinomasand Embryonic Stem Cells: A Practical Approach (E.J.Robertson, Ed.), IRL Press, Oxford,1987). Clonally selected embryonic stem cells are generally more effective in producingtransgenic animals since they have a greater efficiency for differentiating into an animal.Examples of suitable embryonic stem cells are the R1 cell line (Nagy A.. et al, Proc NatlAcad Sci 90:8424â8428, 1993 and Wood et al, Nature 365:87â89, 1993).Pluripotent cells may be obtained from any vertebrate species (i.e. mammals, birds,reptiles, amphibians, and fishes); however, cells derived or isolated from mammals such asrodents (e.g. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle, primates, andhumans are preferred.The fluorescent protein marker may be a Green Fluorescent Protein (GFP) from thejellyfish A. victoria, or a variant thereof that retains its ï¬uorescent properties when expressedin vertebrate cells. Variants of GFP may be selected that have longer wavelengths ofexcitation and emission, are more ubiquitously expressed, have increased thermostability,and/or exhibit stronger ï¬uorescent signals compared to wildâtype GFP. Examples of GFPvariants include a variant of GFP having a Ser65Thr mutation of GFP (S65T) that has longerwavelengths of excitation and emission, 490nm and 510nm, respectively, compared to wild-type GFP (400nm and 475nm); a blue ï¬uorescent variant of GFP (e.g. Y66HâGFP) (Heimet al, Proc. Natl. Acad. Sci. 91:12501, 1994), MmGFP (M. ZernickaâGoetz et al,Development 124:1133-1137, 1997) enhanced GFP (âEGFPâ) (Okabe, M. et al, FEBSLetters 407:313â319, 1997; Clontech, Cal.), or a red shifted variant, EYFP (yellowfluorescent protein; excitation max. 513 nm). Other GFP variants are described on theworldwide web at the Structural Classification of Proteins site(http://www.pdb.bnl.gov/scop/date/ scop1.004.07.001.001.000 html). In an embodiment ofthe invention, the fluorescent protein marker is EGFP which has a Phe to Leu mutation atposition 64 resulting in the increased stability of the protein at 37°C and a Ser to Thrmutation at position 65 resulting in an increased ï¬uorescence. EGFP commercially availablefrom Clontech incorporates a humanized codon usage rendering it âless foreignâ tomammalian transcriptional machinery and ensuring maximal gene expression. The codingsequence of Clontechâs EGFP contains over 190 silent mutations that create a humanizedopen reading frame. Additionally sequences upstream of the EGFP have been converted to1015202530CA 02264450 1999-03-05-12-a Kozak consensus ribosome binding site, allowing for more efficient translation of themRNA in mammalian cells.The pluripotent cells of the invention are produced by introducing into pluripotentcells a nucleic acid sequence encoding a ï¬uorescent protein marker and optionally a nucleicacid sequence encoding a protein of interest, under conditions so that the ï¬uorescent proteinmarker is selectively integrated into a heterologous sex chromosome in the cell.The nucleic acid sequence encoding a ï¬uorescent protein marker may be introducedinto pluripotent cells using an appropriate expression vector which ensures good expressionof the protein. Possible expression vectors include but are not limited to cosmids, plasmids,or modified viruses (e. g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.The invention therefore contemplates an expression vector containing a nucleic acidsequence encoding a ï¬uorescent protein marker, and the necessary regulatory sequences forthe transcription and translation of the inserted proteinâsequence. Selection of appropriateregulatory sequences is dependent on the host cell chosen as discussed below, and may bereadily accomplished by one of ordinary skill in the art. In particular, the vector may containa heterologous or homologous promoter for RNA polymerase H, a downstreampolyadenylation signal, and an enhancer. Suitable promoters include the [3âactin promoter,elongation factor lot, cytomegalovirus promoter, and lac promoter. Suitable enhancersinclude the cytomegalovirus enhancer element. Vectors may encode more than oneï¬uorescent protein marker, or they may encode other reporter proteins. A separate vectormay be used to introduce another ï¬uorescent protein marker or other reporter proteins. Thevectors may be obtained commercially or assembled from the sequences described byconventional methods in the art. In an embodiment of the invention, the commerciallyavailable vectors p EGFP-1, pEGFPâN1, and pEGFP-C1 are utilized.A nucleic acid sequence encoding a GFP or variant thereof may be introduced intoembryonic stem cells via conventional techniques such as calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporationor microinjection. Suitable methods for transforming and transfecting host cells can befound in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory press (1989)), and other laboratory textbooks.A nucleic acid sequence encoding the ï¬uorescent protein marker (e.g. GFP or a1015202530CA 02264450 1999-03-05-13-variant thereof) is introduced into pluripotent cells under conditions so that the ï¬uorescentprotein marker is selectively integrated into a heterologous sex chromosome in the cell. Thepluripotent cells are cultured using conventional methods.The pluripotent cells may be analyzed to corroborate the introduction of aï¬uorescent marker protein such as GFP or a variant thereof. For example, restrictionmapping, Southern hybridization assays, and the like may be performed. The polymerasechain reaction is also useful for this purpose. Appropriate primers may be chosen for thePCR reaction to screen cells for introduction of the vector having a sequence encoding aï¬uorescent protein marker. The primers are chosen to be complimentary to sequences to theï¬uorescent protein marker e. g. GFP or variants thereof.The pluripotent cells of the invention may be used to produce a pluripotent stem cellline using conventional methods. For example, the embryonic stem cells may be coculturedwith feeder cells (usually irradiated fibroblasts or stomal cells e. g. STO cells), or culturedin medium conditioned by established teratocarcinoma stem cell lines and other cells (SeeE.J. Robertson In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach(E.J.Robertson, Ed.), IRL Press, Oxford, 1987). The embryonic stem cells can be propagatedwithout a feeder cell layer in the presence of differentiation inhibiting activity (DIA) or inthe presence of factors such as leukocyte inhibitory factor (Gough, N.M. et al, Reprod. Fertil.Dev. 1281-288, 1989). The factors may be added to the culture or they may be produced bytransforming the stromal cells so that they secrete the factor. The pluripotent cells may alsobe cultured in vitro to produce embryoid bodies.The pluripotent cells of the invention may also be introduced into a blastocyst oraggregated with morula stage embryos to produce chimeric embryos. The chimeric embryosmay be transferred into pseudopregnant recipients, and developed to term to yield transgenicanimals. A transgenic animal of the invention may be used to breed additional animalscarrying a heterologous sex-linked ï¬uorescent marker protein. The transgenic animals of theinvention may also be bred to homozygosity and/or bred with other transgenic animals. Ananimal exhibiting ï¬uorescence and having germline transmission of the marker gene maybe used to establish a transgenic line. A transgenic animal may be mated with a normalanimal, and fertilized embryos collected and cultured.The invention also provides a method of monitoring expression of a protein ofinterest linked to a heterologous sexâchromosome in a nonâhuman animal. The method1015202530CA 02264450 1999-03-05-14-involves introducing into pluripotent cells a transgene comprising a DNA sequence encodinga protein of interest and a sequence encoding a ï¬uorescent protein marker under conditionsso that the ï¬uorescent protein marker is selectively integrated into the heteorlogous sexchromosomes in the cells. The DNA sequences include structural genes (e. g. DNAsequences encoding proteins including introns and exons in the case of eukaryotes),regulatory sequences such as enhancer sequences, promoters, and the like and other regionswithin a genome of interest. The pluripotent cells are cultured in conditions permittingexpression of the ï¬uorescent marker protein and protein of interest. The expression of theprotein is monitored by monitoring the expression of the ï¬uorescent protein marker in thepluripotent cells.The pluripotent cells may be aggregated with an early stage embryo or injected intoa blastocyst as described herein, and the expression of the protein may be monitored bymonitoring expression of the ï¬uorescent protein marker in the chimeric embryo or in thegrowing chimeric embryo. The chimeric embryo may be implanted into a pseudopregnantfoster animal as described herein and the expression of the gene may be monitored bymonitoring expression of the ï¬uorescent protein marker in the growing chimeric embryo orin the resultant transgenic animal.It will be appreciated that a cell, embryo, or transgenic animal of the invention maybe labeled with more than one ï¬uorescent protein marker, or additionally labeled with oneor more other reporter proteins.The invention also provides a method of selecting pluripotent cells and embryoscontaining cells having a preselected heterologous sex chromosome. Pluripotent cellscontaining a heterologous sex-linked ï¬uorescent marker protein may be prepared using themethods described herein and they may be cultured in conditions permitting expression ofthe ï¬uorescent protein marker. Pluripotent cells containing a heterologous sex chromosomeare selected by selecting embryonic stem cells expressing the ï¬uorescent protein marker.Embryos comprising a preselected heterologous sex chromosome produced using themethods described herein, can be selected by selecting embryos expressing the ï¬uorescentprotein marker which is linked to the heterologous sex chromosome. For example, if theï¬uorescent protein marker is GFP, embryos containing the heterologous sex chromosomecan be readily selected using a ï¬uorescence microscope. Selection can be observed at themorula stage or blastocyst stage. Embryos can be monitored at 470nm to 490nm during in1015202530CA 02264450 1999-03-05_ 15 _vitro culture.The methods of selecting embryos containing a preselected heterologous sexchromosome in their cells may be used to sex animals. The methods have application inbreeding rodents for laboratory use, and for breeding farm animals such as bovines, sheep,pigs, goats etc. The methods described herein may be particularly useful in reducing sex-linked genetic disorders in farm animals. The methods may also be used to increaseagricultural production. For example, the bovine dairy and beef sectors require female andmale animals respectively, and the methods of the present invention enable selection offemale animals.The following non-limiting example is illustrative of the present invention:Example 1Materials and MethodsVectors used: pCXâEGFP, the EGFP expressing vector has been previously described (10).It contains EGFP inserted into pCAGGS, a plasmid vector containing a chicken beta-actinpromoter and CMV enhancer, beta-actin intron and rabbit Bâglobin polyadenylation signal17' pPGKPuro, the puromycin resistance gene containing vectorâ was used for co-electroporation since it carries an antibiotic resistance gene.Creating green ES cells: Cells were co-electroporated under standard conditions with 40-80ug of linearised pCX-EGFP (BamHI or Scal used interchangeably for linearisation) and10mg of circular pPGKPuro. The plasmid that is to integrate into the genome is linearised,but the drug selection containing plasmid is used in a circular form since this is unlikely tointegrate thereby conferring transient drug resistance. Each individual electroporation wasseeded onto a single 10mm gelatin coated tissue culture plate and resulted in approximately80-140 puromycin resistant colonies, 8-10 of which were green. Control electroporationswere carried out using one or other of the plasmids singly with subsequent selection inpuromycin. Selection in puromycin was initiated 48 hours after electroporation, andcontinued for 3-4 days thereafter. Colonies were viewed under a dissecting microscope(Leitz MZ8) equipped with a GFP illumination source (produced by Leica) and scored forgreen by etching the underside of the tissue culture plate with a needle. Colonies assignedas being âgreenâ were picked into 96 well plates for expansion and freezing.Production of transgenic mice: Both diploid and tetraploid aggregations were performedto produce embryos using conventional techniques.1015202530CA 02264450 1999-03-05-16-Observation of green ï¬uorescence: The EGFP ï¬uorescence was examined underdissecting microscopes (Leitz MZ8 and MZ12) equipped with GFP excitation sources(Leica). The excitation maxima of EGFP is at 488nm, and the emission maxima is at 507nm,the optics provided by Leica were specifically designed to detect this chromophore. Bothtissue culture dishes and mouse embryos and adults could be viewed under the same optics.Results.Expression of EGFP in ES cells in culture: The pCXâEGFP plasmid (Okabe et al., 1997)was introduced into R1 ES cells via a co-electroporation with a drug selection encodingplasmid. Both neomycin (ploxPneo) and puromycin (pPGKPuro) encoding plasmids wereused in initial trial co-electroporation studies, but eventually solely pPGKPuro was used.The reasons for this being twofold: first any subsequent genome alterations to these parentalgreen cells would most probably incorporate a neo (neomycin) selectable marker as it ismore widely used than puro (puromycin), and second, the killing efficiency of puro withina short period of time (that conferred during transient drug resistance - since the drugresistance encoding plasmid is electroporated in circular form) is greater than that of neo.A total of 10 co-electroporations were carried out with the pPGKPuro selectionplasmid, and 4 with ploxPneo. Only colonies from the pPGKPuro co-electroporations werepicked for expansion and freezing. Differences were observed in the intensity of EGFPexpression in cells in culture. This was first noted when puromycin resistant colonies wereassayed for expression of EGFP prior to their picking into 96 well plates.Identifying ubiquitous expressing green ES cell lines: The initial aim was to establish aline of ES cells that exhibited ubiquitous GFP expression in all embryonic and adult tissues.Even though the promoter/enhancer combination utilized within the pCXâEGFP constructhas been reported to drive strong uniquitous expression in mice, position effects can ofteninï¬uence the expression of a transgene insertion in a locus specific fashion. To this endtetraploid aggregations between the puro restistant green ES cells and wild type tetraploidhost embryos were performed so as to assay the extent of expression within embryos ofseveral lines. Lines were subsequently chosen for diploid aggregations so as to producegermline transmitting chimeras.Chimeric analysis to assay ï¬delity of green cells as a cell autonomous marker: Severalof the lines that were believed to exhibit ubiquitous GFP expression within mouse embryoswere chosen for diploid aggregations that were not taken to term, thereby permitting theobservation of green ES cells in utero and assessing their fidelity as a marker within1015202530CA 02264450 1999-03-05-17-chimeras, i.e. could green ES cells be discerned from wild type ES cells within a mixedpopulation in low to high level chimeric embryos.Generation of F1 mice: Four lines (B4, B5, C1, and D4) exhibited varying degrees ofubiquitous expression in tetraploid aggregation derived embryos, and were thus chosen forthe generation of lines of green mice through dipoid aggregations. Of these lines 3 gavegermline transmitting chimeras, B4 did not. The BS and D4 ES cell line aggregations eachgave one good chimera, and C1 yielded 2. Male chimeras produced by diploid aggregationwere mated with CD1 or ICR female mice, with the resulting Fls being analysed forMendelian inheritance of the trans gene and for the extent of EGFP expression in the pups.These results indicate that lines B5 and C1 contain autosome integrated transgenes (bothsexes of F1 pups randomly inherit the transgene), but D4 contains an Xâlinked transgeneinsertion (only F1 females inherit the transgene from a male chimera - note also that R1 isa male ES cell line). The first two D4 chimera mated females gave birth to a total of 22 F1pups, 11 of which were female, transgene activity (indicated by green newborn pups) wassolely confined to these females. Over 100 pups have been born, with only the femalesexhibiting transgene activity.Interestingly all three lines (B5, C1 and D4) exhibited almost ubiquitous greenexpression in tetraploid embryos, though the actual intensity varied dramatically with C1expressing at the lowest level, and B5 expressing at the highest. Since it was uncertain ifhigh level GFP expressing lines would be deleterious it was decided to use both C1, B5 andD4 for the diploid aggregations. Interestingly of the newborn F1 mice B5 exhibited almostubiquitous green expression, C1 showed more restricted expression (primarily in the CNS),and D4 females exhibited ï¬uorescence in a mosaic fashion indicative of Xâinactivation.Non-invasive sexing of pre-implantation embryos: The Xâlinked transgene insertion inthe D4 EGFP ES cell line allows the sex-specific selection of individuals at any stage (from8 cell stage onwards) of development or adult life. This is the first case where a nonâinvasivemarker can be used for sex selection, being particularly amenable to application during pre-implantation stages of development, thereby creating more efficient and cost effectivebreeding regimes in mice and other mammals.In an ongoing experiment matings have been set up between the D4 chimera andestrous CD1/ICR female mice, E3.5 pre-implantation stage embryos have been ï¬ushed fromthe uterine horns and viewed under GFP optics, in total approximately 50% appear tocontain transgene activity. These blastocysts have been separated into green (prospective1015202530CA 02264450 1999-03-05-13-female) and nonâgreen (prospective male) pools and transfered into foster mothers which areexpected to give birth to single sex pups depending on the colour coded pool of thetransferred embryos. Additionally preimplantation stage embryos will be subjected to PCRprotocol that can detect the presence of a Yâchromosome and so discern between the sexes,thereby confirming that the nonâinvasive method for sexing offspring can be carried out atany stage during development.Example 2Generating Green Fluorescent Mice by Germline Transmission of Green FluorescentES CellsEstablishing lines of green ï¬uorescent ES cells. Embryonic stem (ES) cells represent apluripotent cell type derived from preimplantation stage embryos. They can be propagatedin culture, genetically modified and subsequently reintroduced into animals14=15.Ubiquitously EGFP (Promega Laboratories, Inc. CA) expressing (green ï¬uorescent) ES celllines were established by coâelectroporation of pCX-EGFP16, an EGFP expressing vector,where the EGFP gene is driven by a CMV (cytomegalovirus) immediate early enhancercoupled to the chicken B-actin promoter and first intron17, and pPGKPuro18 a selectablemarker containing vector. Since the majority of targeting cassettes incorporate a neomycinresistance gene, puromycin was chosen as the selectable marker, thereby facilitating a secondin vitro modification event if one is required. Of the surviving puromycin resistant coloniesonly green clones were selected for further investigation. These clones were visualized asgreen ï¬uorescing colonies under a dissecting microscope equipped with ï¬uorescent optics,and picked into 96 well gelatinized tissue culture plates. Clones were grown in duplicatewith one plate being frozen down as a stock. A variation in the intensity and homogeneityof EGFP expression was observed between clones, possibly reï¬ecting the copy number andthe effect of site of integration of the transgene. Figure 1A shows a line of green ï¬uorescentES cells grown on gelatinized tissue culture plates, and Fig. 1B is a puromycin resistantgreen ï¬uorescent colony. In most cases maintenance of the green ï¬uorescence was observedin differentiated ES cells (Fig. 1C), whereas embryoid bodies plated on embryonic feedercells (Fig. 1D) exhibited ubiquitous ï¬uorescence though at varying levels, supportingprevious observations that the promoter used in this study is expressed in all nucleated cellsbut is upregulated in some lineages. Increased ï¬uorescence was consistently observed inendodermal cells situated on the surface of embryoid bodies (Fig. 1D arrows), and suggest1015202530CA 02264450 1999-03-05-19-that these cells may be equivalent to parietal endoderrnal cells which were observed to giveincreased ï¬uorescence over other cell types in transgenic mouse embryos established fromthis ES cell line.Selection criteria for ES cell lines - an in vivo test. In order to produce germlinetransmitting chimeras several different lines of green ï¬uorescent ES cells were tested fordevelopmental potential and ubiquitous embryonic expression by tetraploid embryoâES cellaggregation19(see later section). Four of these lines tested by tetraploid embryoâES cellaggregation were chosen for diploid embryo â ES cell aggregations20 in order to obtaingermline transmitting chimeras.Pre-selecting chimeric embryos for transfer into recipient females. AggregatingES cells with diploid eightâcell stage embryos is a simple and inexpensive means ofintroducing genetic alterations into mice. Circumventing the transfer of nonâchimericembryos after the aggregation is completed makes the technology more efficient and reducesanimal space requirements. The B5/EGFP ES cell line now provides a convenient tool to doso. Figure 2 illustrates different ES cell contributions into morula (Fig 2A) and blastocyst(Fig 2BâF) stage embryos obtained after overnight incubation of eight cell stage embryo-B5/EGFP ES cell aggregates. While ES cells will preferentially contribute to the ICM, wherethey can either represent the entire ICM (Fig 2B) or just a portion of it (Fig 2C, 4D), theywill also occasionally be present in the trophoblast (Fig 2E arrow, and 2F), though it isunclear whether this is a real contribution to the trophoblast lineage or whether these cellsare trapped in this region of the embryo never to proliferate.Germline transmission of the B5/EGFP and C1/EGFP transgene - diploidembryo - ES cell aggregation. Reported below are the results of experiments performedwith two of the four ES cell lines selected for germline transmission, which were exhibitingextremes in expression of the transgene, as assessed by tetraploid aggregation (these beingB5/EGFP a line exhibiting a high level of ubiquitous green ï¬uorescence and Cl/EGFP a lineexhibiting ubiquitous green ï¬uorescence at a low level). They were chosen since no data wasavailable on the viability of homozygous mice expressing EGFP at high levels, and to ensurethat the integration did not disrupt an essential locus. Figure 3A is a schematic illustrationof the diploid aggregation technique in which a single wild type morula stage host embryois aggregated with a clump of green ï¬uorescent ES cells resulting in the preferentialincorporation of the cells into the inner cell mass (ICM) of an embryo by the time it reaches1015202530CA 02264450 1999-03-05-20-the blastocyst stage (after 24 hours of incubation). The sequence in Figures 3B-E shows theprogression of aggregation during overnight incubation. In order to observe the fidelity ofEGFP as a cell autonomous marker within a mosaic population of cells, some of thetransferred blastocysts were recovered at later (postimplantation) stages of embryonicdevelopment. Individual EGFP expressing cells can be identified in chimeric embryos atstages E7.75 (Fig. 4A) and E8.5 (Fig. 4B).It was also noted that in addition to black eyes and pigmented coat (representativeof the 129 mouse strain the R1 ES cells are derived from), chimeras could be identified andassessed for the strength of their ES cell contribution by viewing newborn pups fromaggregation recipients under the appropriate fluorescence optics (Fig. 4C and 4D, show astrong chimera). Male chimeras with a high ES cell contribution were crossed with CD1 orICR females, and F1 mice were genotyped using the appropriate optics. Both the B5/EGFPand Cl/EGFP chimeric mice transmitted the EGFP transgene in a Mendelian fashion.B5/EGFP mice were observed as ubiquitously green throughout embryonic development andadult life (see following section and Fig. 6), whereas in the C1/EGFP line, EGFP wasubiquitously expressed (though at a lower level than in the B5/EGFP line) during early tomidâgestationa1 embryonic development then became restricted to pancreas and certainregions of the CNS in newborns and adults (data not shown). Therefore the data presentedhere will focus on the B5/EGFP line.Expression of the B5/EGFP transgene in preimplantation embryos. The onsetof zygotic transgene expression was assayed for from the twoâcell stage on. No greenfluorescence was observed in 1 to 4âcell stage embryos derived from crosses betweentransgenic males obtained after germline transmission and wild type females (Fig. 5A).Zygotic expression from the B5/EGFP transgene initiates at preimplantation stages ofdevelopment, with green ï¬uorescence first detected as morula stage embryos begin tocompact (Fig. SB), and by the blastocyst stage expression is clearly visible (Fig. 5C and 5D).On the other hand, from the analyses of non-transgenic embryos derived from matingsbetween wild type males and heterozygous females, it was found that residual maternalexpression of the transgene is observed until after implantation (Fig. 5E-5G), suggesting thatthe EGFP transcript or protein is very stable or that the maternal transcript is very abundant.Due to the high level of maternal transgene expression, all preimplantation stage embryosderived from such a cross were observed as being green ï¬uorescent, though only half theembryos inherited the transgene (Fig 5Eâ5G). It was also noticed that green ï¬uorescence1015202530CA 02264450 1999-03-05-21-resulting from maternal expression is more pronounced than zygotic expression up until theblastocyst stage. A comparison of embryos derived from a heterozygous male crossed to awild type female (Fig. 5H left, note that about half the embryos are green), and a wild typemale crossed to a heterozygous female (Fig. 5H right, note that all the embryos are greenthough only half will carry the EGFP transgene) illustrates this observation. The residualmaternal transgene expression observed in preimplantation embryos is lost afterimplantation, probably as a result of the degradation of the EGFP transcripts or protein, andthe rapid increase of the cellular mass that is observed concurrent with implantation at E4.5â4.75, and the onset of gastrulation at E6.5.Expression of the B5/EGFP transgene in postimplantation embryos.Postimplantation expression of the B5/EGFP transgene in embryos and extraembryonictissues appeared to be ubiquitous throughout development (Fig. 6). It was also found that thegreen ï¬uorescence facilitated the location and isolation of postimplantation embryos atearlier postimplantation stages (E4.75-E5 .75) which is otherwise extremely difficult. Thismeans that such a green ï¬uorescent transgenic line allows access to embryos at all stages ofdevelopment; from preimplantation through postimplantation to birth. Fig. 6A shows anE4.75 (late hatched blastocyst) green ï¬uorescent embryo derived from a cross between aB5/EGFP male and a wild type female, the embryo has just implanted and is surrounded bysome residual nonâï¬uorescent uterine tissue. Fig. 6B shows a uterus that has been cut openat the proximal side (opposite the mesometrial side) showing early stage decidual tissuewhich has been torn open to expose an E5 green ï¬uorescent embryo. Figure 6C shows anE5.5 green ï¬uorescent embryo dissected away from the uterus but still surrounded by someresidual decidual tissue. By E9 (Fig. 6D, 6E) it is clearly visible that both the embryo properand extraembryonic tissues express the EGFP transgene. Starting at E9 in theextraembryonic lineage (Fig. 6E), the parietal endoderm exhibits highest levels of trans geneexpression, this observation is supported by the differentiated embryoid body data whereendoderm cells regularly exhibit increased ï¬uorescence (Fig. 1D arrows). At allpostimplantation stages no ï¬uorescence is observed in non-transgenic embryos, as illustratedin Figures 6F and 6G. Figure 6F shows a bright field normal optic view of two E9.5littermates derived from a cross between a transgenic male and a wild type female (only onehas inherited the transgene), with Fig. 6G being a dark field ï¬uorescent optic view whereonly the transgenic embryo can be seen to ï¬uoresce. The residual maternal transgeneexpression observed in preimplantation embryos is thus lost after implantation, probably as1015202530CA 02264450 1999-03-05-22-a result of the rapid cellular proliferation that is observed concurrent with implantation atE4.5â4.75, and the onset of gastrulation at E6.5. After midgestation transgene expression ismaintained in both the embryo and placenta (Fig. 6G). All tissues appear to express thetransgene including the heart and lungs (Fig. 61) in addition to the kidney rudiment andintestine (Fig. 6]). The only cells that appear not to ï¬uoresce green are enucleated cells suchas erythrocytes. Additionally cells and tissues with increased hemoglobin content, such asblood vessels, spleen and liver, exhibit reduced ï¬uorescence as their development proceedssince the hemoglobin masks the green ï¬uorescence.Expression of the B5/EGFP transgene in adult mice. As with transgenic embryos,the B5/EGFP transgene is ubiquitously expressed in newborn (Fig. 7Aâ7C) and adult mice(Fig. 7D-7F). Transgenic newborn pups can be recognized due to ï¬uorescence in the skin(Fig. 7A, 7B), but as pups mature the ï¬uorescence is obscured in the fur coat covered bodyparts, especially if the fur is pigmented (Fig. 7C). This contrast is clearly observed betweenadult mice with different coat colours, and is illustrated here by albino (Fig. 7D and 7E),grey (chinchilla), and agouti (Fig. 7F, tails left to right). The entire organ system of theB5/EGFP transgenic line ï¬uoresces green and, even though ubiquitous, the level ofexpression varies between different organs, with musculature (Fig. 7G), brain (Fig. 7H) andpancreas (Fig. 7K) exhibiting the highest levels of transgene expression, intestine (Fig. 7L)expresses at a lower level. Kidney (Fig. 71) and liver (Fig. 7]) also do not strongly ï¬uoresceas they have a high hemoglobin content.Mouse embryos derived solely from ES cells: wt tetraploid embryo - GFP EScell and GFP tetraploid embryo - wt ES cell aggregations. The aggregation of ES cellswith tetraploid 4âcell stage embryos results in the production of a completely ES cell derivedpostimplantation embryo, and provides a way to study the phenotype of a given ES cell line,circumventing germline transmission19=21a22, or to separate embryonic and extraembryoniccomponents of a compound phenotype resulting from gene ab1ation23. The technique isillustrated in Figure 8A, with Figures 8B-E illustrating the process of aggregation. The toppanel of Figure 9 shows embryos derived from a wild type tetraploid embryo - B5/EGFP EScell aggregation experiment where the embryo will be green ï¬uorescent (Fig. 9A, 9B),whereas the lower panel illustrates the reverse a B5/EGFP â wild-type ES cell aggregationwhere the ectoplacental cone/placenta will be green ï¬uorescent (Fig. 9C, 9D). With simplemicroscopic observation the expected separation of the tetraploid and ES cell compartments1015202530CA 02264450 1999-03-05-23-to the extra embryonic tissues and embryo proper, respectively, could be confirmed.DiscussionThe B5/EGFP line of ES cells represents a source of pluripotent, green ï¬uorescentcells which can be manipulated in vitro by further transgenesis or targeted gene alteration,then reintroduced into mice. Gerrnline transmission of these ES cells has allowed us toestablish that EGFP is ubiquitously expressed in all cellular progeny of the ES cells. TherebyEGFP would provide a mutant cellâautonomous marker for mutant cells which would thenbe coupled to a second genetic alteration. These cells could be used for mutant celltransplantation experiments or chimeric analyses, where the GFP expression provides anexceptional tool to follow the behavior of the mutant cells during development or diseaseprocesses, such as cancer.Alternatively, the transgenic mouse line derived from the âprimaryâ B5/EGFP EScell line can provide a ubiquitous GFP tag for normal cells and tissues for similarexperiments where opposite marking is required. In addition, green ï¬uorescence can be usedas a means to identify and dissect early stage preimplantation embryos at stages (E4.75 â 6.0)that have previously been elusive to all but the most highly skilled researchers. Beside theabove main applications other important uses are envisioned for the GFP tagged ES-cell /transgenic-mouse system. Green ï¬uorescent ES cells should increase the efficiency ofchimera production by allowing the preselection of chimeric blastocysts prior to transfer intorecipient females.Unlike standard transgenic regimes where genotyping is performed by either PCR,Southern analysis or the use of a chromogenic substrate for reporter gene visualization, usingthe present invention transgenic embryos and pups were identified by visualization of greenï¬uorescence. When a litter of pups derived from a cross between an animal heterozygousfor the transgene and a non-transgenic is viewed under the appropriate optics only half theanimals will ï¬uoresce (e. g. Fig. 10). Such a ï¬uorescence based reporter strategy offers theadvantage not only in that transgenic animals are readily and non-invasively identified, butalso that animals homozygous for the transgene can be recognized due to their increasedï¬uorescence over heterozygotes a feature that can only be determined in standard transgenicsby breeding animals or by cloning the site of transgene insertion.The tetraploid embryo â ES cell aggregation provides a fast and inexpensive accessto the embryonic phenotypes of mutations created in ES cells and also generates polarizedchimeras in which the embryo proper, amnion and yolk sac mesoderm is compeletely ES1015202530CA 02264450 1999-03-05-24-cell-derived whereas the yolk sac endoderm and trophoblast lineages are tetraploid embryo-derived (Nagy, A et al, Development 110, 815, 1990). The feasibility of this technology wasdemonstrated in an earlier studyzg. In such studies it is essential to tag the tetraploid cellswith a reporter gene to ensure that the embryos are completely mutant ES cell-derived. Untilnow, the Rosa 26 transgenic 1ine24 providing ubiquitous expression of a lacZ reporter hasbeen the line of choice. Here the superiority of the GFP transgenic mouse line isdemonstrated for tagging the tetraploid compartment of an embryo.Through demonstrating some of the applications of the ubiquitous GFP reportersystem in ES cells and the corresponding transgenic mouse line, the intensively evolvingmouse genetic technologies will incorporate the tool described herein in many aspects of itsmethodologies.Experimental protocolGeneration of green ES cells. pCX-EGFP, an EGFP containing vector has been describedpreviouslylo. It contains EGFP inserted into pCAGGS, a plasmid vector containing achicken beta-actin promoter and CMV enhancer, Beta-actin intron and bovinepolyadenylation signalâ. pPGKPuro, a puromycin resistance gene containing vectorcontaining a PGK promoter18 was used for coâelectroporation since it carries an antibioticresistance.R1 ES ce1ls19 were maintained as described previously25. Cells were co-electroporated under standard conditions25a26 with 40â80].Lg of linearised pCX-EGFP(BamHI or Seal were used interchangeably for linearisation) and 10mg of circularpPGKPuro18. Each individual electroporation was seeded onto a single 10mm gelatin coatedtissue culture plate and resulted in approximately 80-140 puromycin resistant colonies, 8-10of which were green as observed under the appropriate optics. Control electroporations werecarried out using one or other of the plasmids singly with subsequent selection in puromycin.Selection in puromycin was initiated 36-48 hours after electroporation, and continued for3-4 days thereafter. Colonies were viewed under a dissecting microscope (Leitz MZ8)equipped with a GFP illumination source (Leica) and scored for green by etching theunderside of the tissue culture plate with a needle. Colonies assigned as being green werepicked into 96 well plates for expansion and freezing25.Embryoid bodies were obtained by seeding ES cells into bacteriological plates and1015202530CA 02264450 1999-03-05-25-by omitting LIF and B-mercaptoethanol from the culture medium. After approximately 1week these embryoid bodies which had been growing in suspension were replated ontofeeder cell containing plates, where they attached and differentiated further27.Diploid embryo - ES cell aggregations. Diploid aggregations were performed asdescribed previously?-0. A detailed protocol can be obtained on the world wide web onhttp://www.mshri.on.ca/develop/nagy/Diploid/diploid.htm. After overnight incubation withcells in depression plates embryos were either transferred to recipient females (for theproduction of germline transmitting chimeras or postimplantation embryo dissections),seeded onto feeder cell containing plates (for the analysis of blastocyst outgrowths) or placedin M2 medium in depression glass microscope slides (for photography).Germline transmitting male chimeras were crossed with ICR females. Subsequently,the B5/EGFP mice have been maintained on an ICR background.Tetraploid embryo - ES cell aggregations. The technique relies on theelectrofusion of 2-cell stage embryos placed between the electrodes of an electrode chamberslide, resulting in rendering their genome tetraploid. The resulting oneâcell stage embryosare incubated in vitro until they reach the 4âcell stage of development at which time they areused to âsandwichâ a clump of ES cells. This arrangement when incubated overnightpromotes the aggregation of the two embryos and the intervening ES cells resulting in theformation of a blastocyst. Tetraploid aggregations were performed as describedpreviouslyzl. A detailed protocol can be obtained on the world wide web onhttp://www.mshri.on.ca/develop/nagy/Tetraploid/Tetra.htm. After aggregation embryos,were treated exactly as for diploid aggregations.Observation of green ï¬uorescence. Oviducts or uteruses of superovulated femaleswere ï¬ushed for recovery of preimplantation stage embryos, PBS was used if embryos werenot to be used for in vitro culture, otherwise M2 or KSOM media was usedl.Postimplantation embryos were dissected in PBS and viewed under both dark field andbright field light optics. Certain types of plasticware were found to be refractory toï¬uorescence, and glass vessels and microscope slides gave superior clarity. For plasticwareCostar 10cm tissue culture, Fisher 10cm bacteriological, and Falcon 3cm tissue culture andorgan culture were routinely used for viewing cells and embryos under ï¬uorescent optics.For long-term storage and sectioning, embryos and adult tissues were fixed in 4%paraformaldehyde at 4°C for between 30 min to 4 h. Fifty um sections were cut in iceâcold1015202530CA 02264450 1999-03-05-26-PBS using a vibratome. Sections were mounted under coverslips in 1:1 glycerol/PBS andwere stored at 4°C.EGFP ï¬uorescence was examined under dissecting microscopes (Leitz MZ8 andMZ12) or inverted microscopes (Leitz DMRB or DMRXE) equipped with GFP excitationsources and appropriate filters (Leica GFP Plus fluorescence filter set). Photographs ofï¬uorescent subjects were taken on Kodak p16OO film shot at 800 or 1600 ASA. Mice wereroutinely genotyped without magnification using a Volpi fibre-optic light source fitted witha blue excitation filter and viewed through a yellow (520nm) barrier filter (Chroma).Photographs of pups and mothers were taken using a Nikon FE10 50mm camera fitted witha 35-75mm lens, a set of closeâup filters, and a yellow (520nm) barrier filter.Example 3Non-invasive determination of sex prior to morphological gonadal differentiation haslong been sought, but up until now no such assay has been available for widespread use. Forexample in mammals, the embryo develops to a substantially advanced stage within themother, thereby requiring the maternal investment of time and energy. Therefore from manyviewpoints information regarding sex outcome could be beneficial1 if determined atpreimplantation, prior to the establishment of the pregnancy. Several attempts have beenmade to identify or predetermine the sex of animals at an early stage of embryogenesis,including density centrifugation-separation of sperm into Y and X chromosome bearingpopulations, followed by in vitro fertilizationz, specific elimination of preimplantation stagemale embryos by antibodies directed against a Y chromosomeâspecific surface antigen3,measurement of X-linked gene dosage between littermates4, in addition to bar bodystaining5, karyotyping6 and PCR detection for the presence of a Y chromosome7 usingbiopsy samples taken from preimplantation stage embryos. Each of these methods isinvasive, labor intensive, potentially harmful to the embryo and subject to erroneous results.GFP is a protein of the jelly fish Aqueoria victoria whose marking of individual cellsin a heterologous system was first demonstrated in the nematode worm C. elegans 8. It isnow becoming the reporter of choice in many different systems, from simple organisms suchas yeast to vertebrates9. GFP acts as a reporter of gene expression that can be viewed in anonâinvasive manner, since it can be visualized without the use of a substrate by simpleobservation under specific illumination. At present GFP and its mutagenised derivativeslo1015202530CA 02264450 1999-03-05-27-provide the only non-invasive markers of gene expression available for use in any biologicalsystem.In the process of establishing green ï¬uorescent ES cell lines in order to createtransgenic mice ubiquitously expressing this novel reporter, a mouse transgenic line wasgenerated carrying an EGFP (Clontech Laboratoties, Inc.) transgene integrated on the Xchromosome (D4/XEGFP transgene). This line represents the first vertebrate system wheresex can nonâinvasively be determined from approximately 48 hours after fertilization, longbefore any of the morphological landmarks that discriminate between the sexes have beenestablished.Over 500 pups have been born from fathers carrying the EGFP marked Xchromosome, and in all cases transgene activity in the F 1 generation was solely confined tofemales (Fig. 11a). F1 females (hemizygous for the transgene) when crossed to non-transgenic males produce offspring 50% of which harbor the transgene in a nonâsex specificmanner. Transgenic male offspring derived from such a mating, between a transgenic femaleand wild type male, ubiquitously express the transgene in the skin, whereas hemizygousfemales exhibit mosaic expression due to random inactivation of one of their two Xchromosomes (Fig. llb). If a transgenic male is crossed to a hemizygous female 75% ofprogeny carry the transgene, represented by three distinct populations; transgenic malesexhibiting ubiquitous green ï¬uorescence (25% of offspring), hemizygous females exhibitingmosaic green ï¬uorescence (25% of offspring), and homozygous females exhibitingubiquitous green ï¬uorescence (25% of offspring).No discemable phenotype, other than green ï¬uorescence, has been observed in micecarrying the D4/XEGFP transgene in either hemiâ or homozygous form. Interestingly thebanded pattern of green ï¬uorescence, rarely seen to cross the dorsal or ventral midline,observed in the skin of newborn hemizygous female pups, appears to be intriguingly similarto the fur coat coloring mosaicism characteristic of a variety of other animals including tabbycats, and mutations at the Xâlinked Mottled (Mo) locus in mice1 1.The extent of the D4/XEGFP transgene expression has been determined duringembryogenesis. Zygotic expression of the transgene first produces a detectable greenï¬uorescence in all cells of the embryo initiating at approximately embryonic day (E) 2.5-2.75, as morula stage preimplantion embryos begin to compact. Studies on postimplantationembryos, newborn pups and adult mice reveal that the transgene is expressed in all nucleated1015202530CA 02264450 1999-03-05-28-cell types catalogued, with expression being slightly higher in heart than in other tissues(Fig. l2bâc, and Fig. 14d). Additionally in hemizygous females where the transgene israndomly inactivated in approximately 50% of all cells of the embryo proper (starting atE6.5-7.0 around the time of gastrulation), close inspection of embryos and ï¬uorescenceactivated cell sorting (FACS) analysis of dissociated cells from such embryos suggests thattransgene expression is mosaic.In order to further compound the idea that preimplantation stage embryo could benonâinvasively sexed, it was decided to color pool, and therefore single sex pool,preimplantation stage embryos and then to transfer them to recipient surrogate females. Ifthe color coded nonâinvasive sexing was truly representative, then these females would beexpected to give birth to single sex litters, of which the female only would ï¬uoresce green.Matings were set up between D4/XâEGFP transgenic males and wild type femalemice. Preâimplantation stage embryos were ï¬ushed from the uterine horns or oviducts ofpregnant females, and viewed under the appropriate illumination for GFP expression inorder to discern their sex. Embryos were then separated into green ï¬uorescent (prospectivefemale) and non-green ï¬uorescent (prospective male) pools, and transferred topseudopregnant surrogate females (Fig. 13). Approximately 200 preimplantation embryoswere color pooled and transferred in this way, encouragingly no deviations from theexpected single sex litters were observed.Preimplantation and midgestation stage embryos were also subjected to PCR sexing,in order to detect the presence of a Y chromosome, and so discern between the sexes (Fig.14). This PCR based approach is currently the favored protocol used for preimplantationsexing of mammalian embryos. The results indicate that only non-ï¬uorescent embryos showevidence for the presence of a Y chromosome. This invasive result further supports theobservations that the Xâlinked EGFP transgenic line offers a nonâinvasive method for sexingoffspring at any stage during embryonic development.The D4/XEGFP transgenic line is the first reported case where a nonâinvasive markerhas been used for sex selection in any organism before sexual dimorphism makes thediscrimination possible. Such an approach is feasible, and particularly amenable toapplication during preâimplantation stages of development, thereby creating more efficientand cost effective breeding regimes in human controlled sexually reproducing species. In thecase of mice a significant impact of such a preimplantation sex selection on ES cell basedgenetic technology is envisioned. By selecting female host embryos for injecting or1015202530CA 02264450 1999-03-05-29-aggregating with male ES cells, 100% germline transmission of the ES cell genome isguaranteed in fertile male chimeras, since only XY containing cells (ES cells in this case)can undergo sperrniogenesis. In larger mammals such as cattle, where breeding is expensiveand time consuming, costs can be streamlined by such nonâinvasive preimplantation sexselection.MethodsDetails of vectors used. R1 ES cells12 were coâelectroporated under standard conditions13with 4oâ80iig of a linearised EGFP expressing plasmid pCXâEGFP14 and 10mg of a circularantibiotic resistance carrying plasmid, pPGKPuro15. Individual electroporations were seededonto 10cm gelatin coated tissue culture plates. After the appropriate period of antibioticselection 80-140 drug resistant colonies remained, approximately 5% of which were green.Cells were allowed to recover for 2 days prior to initiation of the drug selection.Production of transgenic mice. Diploid aggregations16 were used to transmit theD4/XEGFP transgene to the germline. The protocol currently used is available on the worldwide web on http://www.mshri.on.ca/develop/nagy/Diploid/diploid.htm.Collection and color coded pooling of preimplantation stage embryos. Matings were setup between D4/XEGFP transgenic male mice and wild type ICR or CD1 females. Noon onthe day of plug formation was taken as representing E 0.5. At E2.5 oviduct, and at E3.5uteruses were ï¬ushed with M2 medium. Eight cell stage embryos or blastocysts werecollected and assayed for green ï¬uorescence. Embryos were pooled into green and nonâgreengroups then either used for individual PCR genotyping, or transferred directly to recipientpseudopregnant females using standard procedures17.Observation of EGFP activity. GFP ï¬uorescence was examined under dissectingmicroscopes (Leitz MZ8 and MZl2) equipped with GFP excitation sources (Leica GFPPlus). Both ES cells and mouse embryos were viewed using the same optics. Newborn pupswere observed and photographed on the stage of a dissecting microscope from which thecondenser lens had been removed. They were viewed through a yellow barrier filter(Chroma) which was mounted onto a Nikon FE10 35mm camera, or incorporated into a pairof eyeglasses.PCR analysis. PCR genotyping was carried out on individual preimplantation stage embryosand yolk sacs from postimplantation stage embryos. The presence of a Y chromosome wasassayed for using primers for Sry18 and the presence of an autosomal gene (positive control)10CA 02264450 1999-03-05-30-was assayed using primers for myogenin19. For postimplantation embryos 30 cycles wereroutinely used, whereas for preimplantation stages two rounds of PCR of 25 cycles eachwere used. Annealing temperature was 60°C.While the present invention has been described with reference to what are presentlyconsidered to be the preferred examples, it is to be understood that the invention is notlimited to the disclosed examples. To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit and scope of theappended claims.All publications, patents and patent applications are herein incorporated by referencein their entirety to the same extent as if each individual publication, patent or patentapplication was specifically and individually indicated to be incorporated by reference in itsentirety.1015202530CA 02264450 1999-03-05_ 31 _References.Example 2:1. Hogan, B., Constantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryos,Cold Spring Harbor Laboratory.2. Li, X., Wang, W., and Luï¬dn, T. 1997. Dicistronic LacZ and alkaline phosphatasereporter constructs permit simultaneous histological analysis of expression from multipletransgenes.Biotechniques 23: 874-882.3. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. 1994. Greenï¬uorescent protein as a marker for gene expression. Science 263: 802-805.4. Prasher, D. C. 1995. Using GFP to see the light.Trends Genet . 11: 320-3.5. Li, X., Zhang, G., Ngo, N., Zhao, X., Kain, S. R., and Huang, C. C. 1997. Deletionsof the aequorea Victoria green fluorescent protein define the minimal domain required forï¬uorescence.J . Bio. Chem . 272: 28545-9.6. Brejc, K., Sixma, T. K., Kitts, P. A., Kain, S. R., Tsien, R. Y., Ormo, M., andRemington, S. J. 1997. Structural basis for dual excitation and photoisomerization of theAequorea Victoria green fluorescent protein. Proc. Natl. Acad. Sci. U S A 94: 2306-11.7. Heim, R., and Tsien, R. Y. 1996. Engineering green ï¬uorescent protein for improvedbrightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol . 6:178-82.8. Cormack, B. P., Valdivia, R. H., and Falkow, S. 1996. FACS-optimized mutants ofthe green ï¬uorescent protein (GFP).Gene 173: 33-8.9. Zemicka-Goetz, M., Pines, J ., Ryan, K., Siemering, K. R., Haseloff, J ., Evans, M.J., and Gurdon, J. B. 1996. An indelible lineage marker for Xenopus using a mutated greenï¬uorescent protein. Development 122: 3719-24.10. Okabe, M., lkawa, M., Kominami, K., Nakanishi, T., and Nishimune, Y. 1997."Green mice" as a source of ubiquitous green cells. FEBS Lett. 407: 313-319.11. Zemicka-Goetz, M., Pines, J., McLean Hunter, S., Dixon, J . P. C., Siemeiing, K. R.,Haseloff, J ., and Evans, M. J. 1997. Following cell fate in the living mouse embryo.Development 124: 1133-1137.12. Takada, T., Iida, K., Awaji, T., Itoh, K., Takahashi, R., Shibui, A., Yoshida, K.,Sugano, S., and Tsujimoto, G. 1997. Selective production of transgenic mice using greenï¬uorescent protein as a marker. Nat. Biotechnol. 15: 458-61.1015202530CA 02264450 1999-03-05-32-13. Nagy, A. 1996. Engineering the mouse genome. Mammalian Development (Lonai,P., ed), pp. 339-371, Harwood Academic Publishers, Amsterdam.14. Capecchi, M. R. 1989. The new mouse genetics: altering the genome by genetargeting.Trends Genet. 5: 70-76.15. Rossant, J., and Nagy, A. 1995. Genome engineering: the new mouse genetics. Nat.Med. 1: 592-594.16. Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y., and Okabe,M. 1995. Green ï¬uorescent protein as a marker in transgenic mice. Develop. Growth Dzï¬er.37: 455-459.17. Niwa, H., Yamamura, K., and Miyazaki, J. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene 108: 193-199.18. Tucker, K. L., Beard, C., Dausmann, J ., Jackson-Grusby, L., Laird, P. W., Lei, H.,Li, E., and J aenisch, R. 1996. Germ-line passage is required for establishment of methylationand expression patterns of imprinted but not of nonimprinted genes.Genes Dev. 10: 1008-20.19. Nagy, A., Rossant, J ., Nagy, R., Abramow-Newerly, W., and Roder, J. 1993.Derivation of completely cell culture-derived mice from early-passage embryonic stemcells.Proc. Natl. Acad. Sci. USA 90: 8424-8428.20. Wood, S. A., Pascoe, W. S., Schmidt, C., Kemler, R., Evans, M. J ., and Allen, N. D.1993. Simple and efficient production of embryonic stem cell-embryo chimeras by coculture.Proc. Natl. Acad. Sci. USA 90: 4582-4585.21. Nagy, A., and Rossant, J . 1993. Production of completely ES cellâde1ived fetuses.Gene Targeting: A Practical Approach (Joyner, A., ed), pp. 147-179, [RL Press at OxfordUniversity Press.22. Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Gertsenstein, M., Pawling, J.,Kieckens, L., Vandenhoeck, A., Desclercq, C., Moons, L., Risau, W., Collen, D., and Nagy,A. 1996. Heterozygous VEGF deficiency causes abnormal vasculogenesis in completelyembryonic stem cell deiived mouse embryos. Nature 380: 435-439.23. Duncan, S. A., Nagy, A., and Chan, W. 1997. Murine gastrulation requires HNF-4regulated gene expression in the visceral endoderrn: tetraploid rescue of Hnf-4'/â embryos.Development 124: 279-287.24. Friedrich, G., and Soriano, P. 1991. Promoter traps in embryonic stem cells: agenetic screen to identify and mutate developmental genes in mice.Genes Dev. 5: 1513-10202530CA 02264450 1999-03-05-33-1523.25. Pirity, M., Hadjantonakis, A.âK., and Nagy, A. 1998. Embryonic stem cells, creatingtransgenic animals. Cell Culture for Cell and Molecular Biologists (Mather, J. P. andBarnes, D. eds) V0]. (in press), Academic Press, San Diego.26. Wurst, W., and Joyner, A. 1993. Production of targeted embryonic stem cell clones.Gene Targeting: A Practical Approach (Joyner, A., ed), pp. 33-62, IRL Press at OxfordUniversity Press.27. Martin, G. R., and Evans, M. J. 1975. Differentiation of clonal lines ofteratocarcinoma cells: formation of embryoid bodies in vitro.Proc. Natl. Acad. Sci. U S A72: 1441-5.Example 3:1. Hare, W.C. Dev Biol 4, 195-216 (1986).2. Hagele, W.C., Hare, W.C., Singh, E.L., Grylls, J.L. & Abt, D.A. Can J Comp Med 48,294-8 (1984).3. Utsumi, K., Satoh, E. & Iritani, A. JExp Zool 260, 99-105 (1991).4. Monk, M. & Handyside, A.H. JReprod Fertil 82, 365-8 (1988).5. Gardner, R.L. & Edwards, R.G. Nature 218, 346-9 (1968).6. Bacchus, C. & Buselmaier, W. Hum Genet 80, 333-6 (1988).7. Gimenez, C., Egozcue, J. & Vidal, F. Hum Reprod 9, 2145-9 (1994).8. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. & Prasher, D.C. Science 263, 802-805(1994).9. Prasher, D.C. Trends Genet 11, 320-3 (1995).10. Heim, R. & Tsien, R.Y. Curr Biol 6, 178-82 (1996).11. Lyon, M.F. Nature 190, 372-373 (1961).12. Nagy, A., Rossant, J ., Nagy, R., Abramow-Newerly, W. & Roder, J . Proc. Natl. Acad.Sci. USA 90, 8424-8428 (1993).13. Pirity, M., Hadjantonakis, A.âK. & Nagy, A. in Cell Culture for Cell and MolecularBiologists (eds. Mather, J .P. & Barnes, D.) 279-293 (Academic Press, San Diego, 1998).14. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. FEBS Lett. 407,313-319 (1997).15. Tucker, K.L., et al. Genes Dev 10, 1008-20 (1996).CA 02264450 1999-03-05-34-16. Wood, S.A., Allen, N.D., Rossant, J., Auerbach, A. & Nagy, A. Nature, , 365, 87-89(1993).17. Hogan, B., Beddington, R., Constantini, F. & Lacy, E. Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory, 1994).18. Gubbay, J., et al. Nature 346, 245-50 (1990).19. Wright, W.E., Sassoon, D.A. & Lin, V.K. Cell 56, 607-17 (1989).1015202530CA 02264450 1999-03-05-35-Figure Legends:Figure 1.Expression of EGFP in undifferentiated and differentiated ES cells. (A) ES cells and(B) drug resistant colonies, exhibiting ubiquitous EGFP expression. (C) differentiated EScells and (D) embryoid bodies also exhibit ubiquitous transgene activity, though the latterdo show differential levels of expression with the surface endoderm layer often containinga population of highly ï¬uorescent cells (indicated by an arrow).Figure 2.Preselection of embryos with a good ICM contribution for transfer into recipientfemales. (A) ES cells colonizing part of (right) and the majority of (left) morula stageembryos. (B) ES cells colonizing most of the ICM but not the trophectoderrn of a blastocyststage embryo. (C,D) ES cells colonizing a proportion of the inner cell mass (ICM) of ablastocyst. (E) ES cells localized to the majority of the ICM with a single cell (arrowed) inthe trophectoderm (tro) of a blastocyst, and (F) ES cells localized to part of the ICM withtwo cells seen in the trophectoderm.Figure 3.Diploid green ES cell - wild type embryo aggregations. (A) schematic diagram detailingthe technique. (B 1-B3) aggregation of a clump of green ï¬uorescent ES cells with a wild typeembryo results in blastocyst formation after overnight incubation, with preferentialcontribution of the ES cells to the inner cell mass (B4).Figure 4.Postimplantation embryos made by diploid embryo - ES cell aggregations. Embryosexhibit mosaic expression of EGFP reï¬ecting their chimerism between wild type (nonâfluorescing) and B5/EGFP ES cell contributions. (A) ventral view of a lateheadfold/presomite stage (E8) embryo, anterior is top, (B) lateral view of an early somitestage (E8.5) embryo, anterior is left, (C) dorsal view of an E8.5 embryo, anterior is left, (D)lateral view of the head and forelimb of a newborn chimera, (E) view of the hindlimbs andtail of a newborn chimera, anterior is up.1015202530CA 02264450 1999-03-05-36-Figure 5.B5/EGFP expression in preimplantation embryos. All embryos in panels AâD are derivedfrom a cross between a male that is heterozygous for the transgene and wild type females,whereas embryos in panels E-H are derived from a cross between a wild type male and afemale heterozygous for the trans gene. (A) 1 - 4âcell stage embryos exhibiting no transgeneexpression, (B) embryos initiate expression at the morula stage (~E2.5). By the blastocyststage expression of the transgene is clearly visible, (C) dark field and (D) bright field viewof E3.5 embryos. Residual transgene expression due to maternal transcripts is observed untilimplantation stages, 2âcell and morula stage green ï¬uorescent embryos bright field (E), darkfield (F), dark field of blastocyst stage embryos (G). At E2.5 ï¬uorescence due to maternaltranscripts (right) is stronger than zygotic (left).Figure 6.B5/EGFP expression in postimplantation embryos. All embryos derived from a B5 malecrossed with ICR female. (A) E4.75 newly hatched and implanted blastocyst attached todecidual tissue (dec), (B) E5, (C) E5.5, (D) E9 embryo with associated ectoplacental cone(EPC) partially contained within the deciduum, (E) E9 extraembryonic membranes, (F),bright field view of two E9.5 litterinates, (G), dark field view of the same two embryos, (H)Ell.5 embryo with placenta, (I) Ell.5 heart and lungs, (J) El1.5 kidney rudiment andintestine.Figure 7.Ubiquitous green ï¬uorescence in B5/EGFP mice and organs. (A) head and forelimb viewof a newborn B5/EGFP heterozygote pup among non-transgenic littermates, head transgenicright and non-transgenic literate left, (B) newborn B5/EGFP heterozygote transgenic (right)and non-transgenic literate (left), (C) 1 week old pups transgenic on the right non-transgenicliterate left, (D) 3 week old albino B5/EGFP mouse anterior view, (E) 3 week old albinoB/EGFP mouse posterior view, (F) posterior view highlighting the tails of two month oldalbino (left), grey (middle), agouti (right) mice illustrating pigment dependent ï¬uorescence,(G) dorsal view of an incision made in 3 week old B5/EGFP mouse body with skin removedto reveal ï¬uorescence in the body. Organs from a 3 week old transgenic mouse right/top andnon-transgenic littermate left/bottom; (H) brain, (I) kidney, (J) liver, (K) pancreas, (L)intestine.1015202530CA 02264450 1999-03-05-37-Figure 8.Tetraploid aggregations to produce completely B5 ES cell derived embryos. (A)schematic diagram detailing the tetraploid technique. (BâE) the progression of anaggregation between green ES cells <-> wild type embryo to the blastocyst stage.Figure 9.Postimplantation embryos made by tetraploid aggregation. (A,B) embryos made by B5ES cell - wild type tetraploid embryo aggregation. (C,D) embryos made by wild type ES cell- B5 embryo aggregation. (A) green fluorescent E9 embryo with non-ï¬uorescentextraembryonic membranes (outline by a hatched line), (B) green fluorescent E11 embryowith mosaic yolk sac and nonâï¬uorescent placenta, (C) E8.5 embryo with green ï¬uorescentextraembryonic membranes, (D) E11 embryo with mosaic yolk sac and ï¬uorescent placenta.Figure 10.Non-invasive genotyping of embryos and adults by U.V. - Mendelian inheritance of theB5/EGFP transgene. Genotyping heterozygotes vs nonâtransgenic animals: (A) newbornpups from a cross between a B5/EGFP heterozygous male and an ICR female, between everyï¬uorescent (transgenic) pup is a non-ï¬uorescent nonâtransgenic animal (indicated by anasteiix). Genotyping heterozygotes vs homozygotes: (B) mice from a litter of pups derivedfrom an intercross between a male and female both of which where heterozygous for thetransgene (homozygotes are highlighted by an asterix), (C) a close view of pupsheterozygous (left) and homozygous (right) for the transgene, (D) tail tips from Ell.5embryos homozygous (top), heterozygous (middle) and nonâtransgenic (bottom).Figure 11. Sex-specific GFP expression in newborn pups. A litter of F1 offspring (malesleft, females right) derived from a cross between a D4/XEGFP transgenic male and a wildtype female (a). 6 pups from a litter derived from a cross between a hemizygous female andwild type male (b). Three phenotypes are observed, nonâtransgenics that do not show anygreen ï¬uorescence (male in the first cross, male and female from the second cross),hemizygous females that exhibit mosaic ï¬uorescence in the skin, and transgenic males thatexhibit homogenous ï¬uorescence. Homozygous females though not shown, arephenotypically identical to transgenic males in being homogeneously ï¬uorescencent.101520CA 02264450 1999-03-05-33-Figure 12. Expression of the D4/XEGFP transgene in a sex-specific manner inpostimplantation stage embryos prior to sexual morphogenesis. F1 embryos at E6.5 (a), E8.5(b), and E10.5 (c), derived from matings between transgenic males and wild type femalesare shown. Non-transgenic males, on the left, can clearly be discerned from hemizygousfemales, on the right. Close visual inspection of the female embryos, or analysis ofdissociated cells from them reveals that the observed green ï¬uorescence is in fact mosaic.Figure 13. Preimplantation stage transgene expression allows sex-specific pooling. A poolof three litters of E3.5 embryos derived from natural matings between transgenic males andwild type females in bright field (a), dark field with background illumination (b), and fulldark field (c), where green ï¬uorescent embryos are female and the nonâflluorescent embryosare male (b). Color and sex pooled embryos (d), which gave rise to single sex litters aftertransfer to surrogate females.Figure 14. Invasive PCR sexing of preimplantation, and midgestation embryos. Bright field(a) and dark field view (b) of a litter of E35 preimplantation stage embryos obtained froma mating between a D4/XEGFP transgenic male and a superovulated wild type female. Adark field view (with background illumination) of a litter of E10 embryos obtained from amating between a D4/XEGFP transgenic male and a wild type female (c). PCR genotypingthe E3.5 (d) and E10 (e) embryos for Sry and myogenin.
