Note: Descriptions are shown in the official language in which they were submitted.
TR~NSGENIC ANIMALS FOR TESTING M~LTIDRVG RESIST~NCE
The present invention is rela~ed generally to
genetic manipulation of organisms. More particularly/
the present invention is related to the production of
transgenic animals suitable for testing in vivo the
utility of khe expression of the multidrug resistance
gene and for the development of novel chemotherapeutic
agents against cancers~
BACKGROUND OF THE INV~NTION
Intrinsic and acquired resistance to multiple
chemotherapeutic agents is a ma~or clinical p~oblem in
the treatment of cancer. Cell lines resistant to multi-
ple drugs such as Vinca alkaloids, doxorubicin (Adria-
mycin), colchicine and actinomycin D have been studied,
including lines derived from a human KB carcinoma cell
line after selection in culture for resistance to a sin-
gle agent (Akiyama~ et al, 1985, Somat. Cell Mol~ Genet.
_:117-126). One human gene responsible ox multidrug
resistance, termed MDR1, encodes a 4.5 kb MRNA which is
elevated in highly multidrug-resistant cell lines, in
some normal tissues and many tumors (Fojo, et al, 1987,
Proc. Natl. Acad. Sci. US~ 84:265-269). These tumors are
from intrinsically drug resistant cancers of the colon,
adrenal, and kidn~y, as well as tumors that had acquired
drug resistance after chemotherapy. The protein product
of the MDR1 gene is a 170 kD membrane glycoprotein (P-
glycoprotein), which is overexpressed in multidrug-
resistant cell lines and acts as a pump to transport
chemotherapeutic drugs out of the cell. As expected for
a druy efflux pump, P-glycoprotein is located in the
plasma membrane of resistant cells, binds both cytotoxic
drugs and ATP and, as indicated by sequence analysis, has
12 membrane-spanning domains (Chen et al, 1986, Cell,
47:3al-389).
It has also been reported that full-length
cloned human MDR1 or mouse mdr cDNAs can confer multidrug
resistance on mouse and human drug sensiti~e cells after
transfection or infection with retroviral vectors (Gros,
et al, 1986, Nature 323:728-731. Ueda, et al, 1987,
Proc. Natl. Acad. Sci. USA 84;3004-3008). However, it
has not heretofore been demonstrated by actual experi-
mental work that acquired drug resistance can result from
somatic expression of the human MDRl gene in _ivo through
genetic manipulation of the embryo or the germ plasm.
SUMMARY OF THE INYENTION
It is, therefore, an object of the present
invention to provide transgenic animals carrying and
expressing the human MDR1 gene.
It is another object of the present invention
to provide an animal model for testing the efficacy of
high degree chemotherapy against tumors.
Other objects and advantages will become evi-
dent from the following detailed description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects, features and many of
the attendant advantages of the invention will be better
understood upon a reading of the following detailed
description when considered in connection with the
accompanying drawings wherein:
Figures lA and lB are a schematic representa-
tion of the construction of expression vectors pHGl and
pHG2.
Figure lA is a construction of pHG1 and isola-
tion of the pronuclear injected fragment.
Figure lB is a construction of pHG2. Letters
indicate restriction enzymes used for cloning and orien-
tation characterization: B, Bam~Il; E, EcoRI; N, NdeI; S,
SalI; X, XhoI. Restriction enzyme sites within parenthe-
sis indicate those sites destroyed after ligation.
Figures 2A and 2B show the results of Southern
hybridization of tail genomic DNA from mice.
Figure 2A is a diagrammatic representation of
the B-actin promoter-MDRl fusion transgene integrated in
the mouse genome. A 3.1 kb labeled fragment is expected
~r~
after EcoRI digestion of a mouse genomic DNA and hybridi-
zation with the 5A probe, derived from the middle part of
MDR1 cDNA, if the transgene is integrated. Symbols and
letters of restriction sites are the same as descri~ed in
Figure 1.
Figure 2B is a Southern blot analysis of
genomic DNA (20 ug) digested with EcoRI and hybridized
with the 5A probe under conditions of low strir.gency
(Lanes 1-5) and under conditions of high stringency
(Lanes 6-10). Genomic DNA isolated from: KB-3-1 cells
(Lanes 1,6); Normal mouse mixed with 10 pg of injected
DNA fragment (Lanes 2,7); Normal mouse (Lanes 3,8);
Negative mouse from a litter produced after pronuclear
injection (Lanes 4,9); transgenic mouse from the same
litter (Lanes 5,10).
Figures 3A and 3B show blot-hybridization
analyses of DNA from founder mice carrying the MDR1
transgene.
Figure 3A is a DNA slot blot analysis of 10 ug
tail DNA, denatured and hybridized with 5A probe at high
stringency condition. NM, normal mouse; M39~M168, trans-
genic founder mice; 1 C or 10 C, indicate copy number of
the transgene as explained in Figure 2B for lanes 2 and
7.
Figure 2B is a Southern blot analysis of tail
DNA from founder mice described in Figure 3A. Hybridiza-
tion was performed with 5A probe at conditions of high
stringency. The arrowhead points to the 3.1 kb fragment;
the arrow shows the fragment of a rearranged product of
the transgene.
Figures 4A and 4B show the inheritance of the
MDRl transgene in mice generated from founder M39 (Figure
3).
Figure 4A is a pedigree of M39 and descendant
mice. Squares, males; circles, females; half-filled
symbols, carrier mice; open symbols, noncarriers.
Figure 4B is a Southern blot analysis of tail
DNA from F2 genera~ion mice. Mou~e numbers correspond to
numbers shown in the pedigree above. Molecular weight
standards (1 kb ladder) (BRL) are indicated.
Figure 4C is a slot blot analysis of tail DNA
from mouse 39 and from its F1 and F2 generation
progeny. Pxobe and hybridization conditions are the same
as described in Figures 3A and 3B.
Figure 5 shows the pedigree expression analyses
of the MDR-39 mouse line. 5quares, males; circles,
females; open symbols, noncarrier mice; half-filled
symbols at bottom, heterozygotes; completely-filled
symbols homozygotes, half-strippled symbols at top, mice
express.ing the transgene as detected by RNA studies;
half-hatched symbols at top, mice expressing the trans-
gene as detected by protein immunofluorescence localiza-
tion. *, mice expressing the transgene in spleen but not
in bone marrow. Mice marked by letters are mice for
which RNA studies are represented in Figure 6.
Eigure 6 shows the results of R~A expression
analysis of normal and transgenic mice. A slot blot of
total RNA samples (10 ug) extracted from bone marrow and
spleen of mice A-~ (as indicated in Figure 5) was hybri-
dized with MDR1 5A probe (left) and humanY-actin probe
(right) at conditions of high stringency and at condi-
tions of low stringency, respectively. 3-1, parental
drug-sensitive KB cell line; 8-5 and V-1, multidrug
resistant sublines; BM, bone marrcw; SP, spleen.
Figure 7 demonstrates the tissue spec.ificity of
the transgene expression.
Figure 7A is a simplified rastriction map of
MDR1 cDNA and the probes used. E, EcoRI; P, YW II.
Figure 7B is a blot hybridization of total RNA
(10 ug) extracted from: Upper: 1, KB-8-5 drug resistant
cell line; 2, KB-3-1 drug sensitive and KB-V-l drug
resistant cell lines; Lower: 1, MDR1 non-carrier mouse;
2, MDR1 carrier mouse. Li, liver; Ki kidney; Lu, lung;
Ov, ovary; Mu, skeletal muscle; BM, bone marrow; Sp,
spleen; He, heart; sr, brain. The same blot was hybri-
dized with different probes, as indicated.
Figures 8A, 8B, 8C and 8D evidences immuno-
fluorescence localization of human P170 in bone marrow
cells from an MDRl transgenic mouse. Bone marrow samples
from either an MDR1 transgenic mouse (Figures 8A, 8B and
8C) or a normal sibling (Figure 8D) were smeared on glass
slides, air dried, and then fixed in formaldehyde. The
smears were labeled using monoclonal antibody MRK15
~anti-human P170) and indirectly labeled with
rhodamine. Equal time exposures show bright expression
of P170 in all cells from the MD~ mouse (Figures 8A', 8B'
and 8C') but not in cells from the control mouse (Figure
8D'). (Figures 8A, 8B, 8C and 8D) represent phase con-
trast images of the cells shown in (Figures A', 8B', 8C'
and 8D'). (Mags = X 630; bar = 6.5 m).
DETAILED DESCRIPTION OF THE INVENTION
~ he above and various other objects and advan-
tages of the present invention are achieved by transgenic
animals carrying and expressing the human MDR1 gene.
Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art
to which this invention belongs. Although any methods
and materials similar or equivalent to -those described
herein can be used in the practice or testing of the
present invention, the preferred methods and materials
are now described.
` Unless mentioned
otherwise, the techniques employed herein are standard
methodologies well known to one of ordinary skill in the
art.
As a first step toward producing the transgenic
animals, a cloned human MDR1 cDNA sequence which contains
the ~DR1 translated sequences as well as the endogenous
3' polyadenylation signal under control of the B-actin
promoter was constructed~
~. ~
~r~
Although there is only one functional s-actin
gene per haploid genome both in human and mouse, B-actin
is one of the most abundant proteins in eucaryotic cells
and is expressed in a variety of tissues, irrespecti~e of
em~ryonic origin. Since B-actin is abundantly expressed
in all cells and evolutionarily conserved, it was rea-
soned that the B-actin promoter may satisfy both require-
ments: high level expression and activity in a wide
range of cell types including drug-sensi~ive cells. The
following materials and methods now exemplify the various
steps in producing and testing of the MDRl expressing
transgenic mice in accordance with the present invention.
M~TERIALS AND METHODS
Materials
Restriction endonucleases and T4 DNA ligase
were purchased from New England Biolabs or Bethesda
Research Laboratories, Inc., and used under conditions
recommended by the supplier. Agarose (Seakem, GTG and
Se Plaque, LMB) was supplied by FMC (Rockland, ME).
Reagents for PAGE were from Bio-Rad. Colchicine was
purchased from Sigma chemicals. All other chemicals were
of analytical grade. Cell culture media were obtained
from Gibco and sera from M.A. Bioproducts, Inc. [p32]_
dCTP and nicX-translation kit were purchased from
Amersham or New England Nuclear.
Bacterial strains, plasmids, cell_lines and mouse lines
E. coli strain D~I5 was used for transforma-
tion. pMDR2000XS which carries the full-length MDR-1
cDNA with a unique XhoI site at the 3' end, was con-
structed in this laboratory (Pastan, et al, 1988, Proc.
Natl. Acad. Sci. USA 85:4486-4490). The pGEM2 vector was
from Promega Biotech ~Madison, WI). pBA-CAT which
carries the bacterial CAT gene under control of the
chicken B-actin promoter in pUC18 was a gift from Dr. B.
Paterson~ NIH 3T3 and KB-3-1 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine sexum and calf serum, respectively. Mouse
'~0:~1.2~
lines RCNIH and B6SJL F were obtained from Jack~on
Laboratories (Bar Harbor, ME).
Plasmid construction
A plasmid for the expression of the MDRl cDNA
under the control of a chicken B-actin promoter was con-
structed as shown in Figure lA. pBA-CAT which carries a
chicken B-actin promoter was cut with SalI and a 0.33 kb
fragment containing the essential B-actin promoter
sequences was eluted after electrophoresis on an agarose
gel. The resulting fragment was inserted into the unique
SalI site of pMDR2000XS, which carries the full-length
MDR1 cDNA downstream from the T7 promoter in a pGEM2
vector (Pastan, et al, 1988, Proc. Natl. Acad. Sci. USA
85:4486-4490). The resulting plasmid has a 330 base pair
_-actin promoter in two orientations at the 5' end of the
MDR1 cDNA. Restriction analysis with XhoI/BamHI was used
to identify a plasmid with the B-actin promoter in the
proper orientation with respect to the MDRl cDNA. The
resulting plasmid ~ermed pHG-1 (pBAP-MDR) was used for
microinjection. Since pHG-l wa~ digested with XhoI
before microinjection (see below)~ the effect oi truncat-
ing the actin promoter with XhoI was determined. Accord-
ingly, pHG-2 (tBAP-MDR) was constructed from pHG-1 as
shown in Figure lB, to tes~ the expression le~el of MDR1
under the control of a truncated B-actin promoter.
pHGl was cut with XhoI and the resulting 4.8 kb
and 2.9 kb fragmen~s were eluted after separation on an
agarose gel. The 2.9 kb fragment containing 60 base
pairs of the 5' region of the B-actin promo-ter was
cleaved with SalI to remove these 60 bp sequences,
dephosphorylated with alkaline phosphatase and ligated to
the 4.8 kb fragment which contains the MDR1 cDNA down-
stream -from the XhoI-SalI (270 bp) truncated promoter.
The resulting plasmid had B-actin promoter-MDRl sequences
in two orientations at the 5' end of the multiple cloning
sites of the pGEM2 vector sequences. Restriction
analysis with XhoI/NdeI was used to identify a plasmid
-- 8
with the truncated B-actin promoter-MDR1 sequence in the
proper orientation with respect to the T7 promoter of the
pGEM2 vector. The resulting plasmid, pHG2 (tBAP-MDR) as
well as pHG1 wera examined for their size and expression
of the MDRl cDNA in human KB-3-1 and mouse NIH 3T3 trans-
fectant cells.
Cell culture and transfection
NIH 3T3 mouse cells and human KB-3-1 cells were
cultured and transfected by a calcium phosphate precipi-
tation method as described by Shen et al, 1986, Mol. Cell
Biol. 6:4039-4044.
DNA preparation for microiniection
pHG1 was cut with XhoI and a 4.7 kb fragment
with MDR1 downstream from the B-actin promoter was
separated from the vector fragment by electrophoresis on
a low melting point agarose gel (See "Plaque" infra).
The ethidium bromide-stained band containing the BAP-MDR1
was excised from the gel and was melted in TE (10 mM
Tris-HC1 ph 8.0, 1 mM EDTA) equilibrated phenol solution
at 70C. Then, 3 volumes of buffer containing 0.25 M
NaC1, TE were added and incubated at 70C for 10 min and
at 40C for 10 min. The aqueous phase was separated,
extracted in chloroform (1:1 v/v) and ethanol precipi-
tated. The DNA pellet was dissolved in 0.2 M NaCl, TE
and purified on a NACS column (Bethesda, Research ~abs)
under conditions recommended by the supplier except that
the DNA was eluted with 1.0 M NaC1, TE, 1~ caffeine. The
eluant was ethanol precipitated to remove excess salt,
washed with 75% ethanol and 95% ethanol and the dried DNA
pellet was dissolved in sterile H20. The DNA sample was
microdialyzed on a Millipore filter (#VMWP01300) against
sterile H2O for 30 min. The sample was removed from the
filter, centrifuged in an Eppendorf centrifuge for 15 min
and 2/3 volume from the top was collected. The concen-
tration of the DNA sample was measured by gel electro-
phoresis uslng ethidium bromide stained DNA standards
(A DNA/Hind III fragments). Finally, the DNA was diluted
*Trademark
9 : r
with microin~ection buffer containing 7.3 mM PIPES pH
7.4, 0.1 mM EDTA, 5 mM NaC1.
Microinjection into fertilized mouse egqs
Fertilized eggs were flushed from oviducts of
B6SJL F1, females and were microinjected with 1-2 ng of
DNA. The microinjected embryos were transferred to CR
NIH surrogate females. Manipulation oE mice and eggs and
the microinjection techniques were done as described by
Hogan, et al, 1986, Manipulating the mouse embryo: A
laboratory manual. Cold Spring Harbor Labora-tory. Cold
Spring Harbor, NY.
Preparation and analysis of mouse genomic DNA
High molecular weight genomlc DN~ was isolated
from 1-2 cm tail samples by standard procedures. 20 ug
of DNA samples digested with EcoRI were electrophoresed
on 1% agarose in lX TBE buffer and transferred from the
gel to nitrocellulose paper by the msthod of Southern
(Southern, 1975, J. Mol. Biol. 98:503-517). The RNA was
electrophoresed in 1% agarose/6% formaldehyde gels as
described by Shen et al, 1986, Science 232:643-645. 10
ug of total RN~ was loaded per lane. Only samples in
which the ribosomal R~A appeared intact were analyzed.
RNA was transferred to nitrocellulose paper or Nytran
membrane (Schleicher and Schuell, Inc.~ as described by
Maniatis, et al, 1982. Molecular cloning: A laboratory
manual. Cold Spring ~Iarbor Labora-tory, Cold Spxing
Harbor, NY. For semiquantitative analysis, RNA samples
were applied to filters by using a slot blot apparatus as
described by Fojo, et al, 1987, Proc. Natl. Acad. Sci.
USA 84:265-269. Hybridization was done as described for
DNA hybridization. Northern and slot blot filters were
then washed in 0.2 x SSC 0.1% SDS at 50C (low strin-
gency) or at 0.1 x SSC, 0.1% SDS at 70C (high strin-
~ency) as indicated in text.
MDR1 hybridization probes
The MDRl probes used were obtained from con-
tiguous areas of an MDR1 cDNA and cover around 85~ of the
-- 10 --
total message. A simplified cDNA map is shown in Figure
7A. Probe 10, a 1.6 kb fragment, was obtained by Eco~I
digest of pMDR10, probe 5A, a 1.4 kb fragm~nt, was
obtained by EcoRI digest of pMDR SA; and probe PW 2 #6,
was obtained by purifying the 0.84 kb fragment from a PW
II digest of pMDR2000 (Uedal et al, 1987, Proc. Natl.
Acad. Sci. USA 84:3004-3008).
Immunofluorescence locali~ation
Bone marrow cells were smeared onto glass
slides, air-dried, then fixed in 3.7% formaldehyde in
phosphate buffered saline for 5 min., at 23C. The
slides from control or transgenic mice were then incu-
bated with mouse monoclonal antibody MRK16 followed by
rhodamine-conjugated affinity-purified goat anti-mouse
IgG as described by Willingham et al, 1987, J. Histochem.
Cytochem. 35:1451-1456.
R~SULTS
Construction of BAP-MDRl plasmids
As described herein above, a chicken B-actin
promoter fragment was inserted 5' to MDR1 cD~A in a
unique SalI site of a human MDR1 cDNA clone (Figure
lA). The resulting plasmid (BAP-MDR) contains 330 bp of
the B-actin promoter sequences followed by 4380 bp of the
MDR1 cDNA, extending from position -140 (SacI site) to
position +4240 (EcoRI site). A 4690 fragment was cleaved
with XhoI and used for the microinjection studies. This
fragment does not include vector sequences, except for a
24 bp region of the multiple cloning site from pGEM2 in
the junction of B-actin promoter, which are followed by
140 bp of the 5' untranslated region of MD~l cDNA, the
full-length MDRl translated sequences, and the 3'
untranslated MDRl sequences including the endogenous
polyA addition signal sequences. However, 60 bp of the
5' end of the B-actin promoter do not exist in ~he micro-
injected fragment. Although the remaining 270 bp of B-
actin promoter sequences include the consensus CAAT and
TATA boxes, it was not known if this truncated fragment
Z(~ .2~
would express MDRl. Thus, an expression vector contain-
ing the MDRl cDNA downstream from this truncated promoter
(tBAP-MDR), but identical in all other respects, was
constructed (Figure lB), to test its expression of MDR1.
Transfection studies
To test the expression level of MDR1 under the
control of the B-actin promoter, as well as the function
of the truncated B~actin promoter, the expression
plasmids pHGl and pHG2 were stably transfected into drug-
sensitive KB-3-1 cells (Table I). pHaMDR, a retroviral
expression vector containing two Ha-MSV LTRs was used as
a positive control. For a negative control, cells were
treated by the same transfection protocol in the absence
of DN~. The same negative results were also obtained
after transfection using vector DNA without MDRl
sequences (data not shown).
Dishes of KB-3-1 cells (1 x 105 cells per 10 cm
dish) were transfected with 10 ug of either pHGl, pHG2,
or pHaMDR by the calcium-phosphate precipitation method
and selected with various concentrations of colchicine
(5-8 ng/ml, which i5 3-5 times as high as the LD50 f
colchicine for the paren~al KB-3-1 cells) for 12 days.
The experiments summarized in Table I show comparable
results for pBAP-MDR and pHaMDR in the number of
resistant colonies at 5 ng/ml of colchicine. However,
the average colony size was smaller for the pBAP-MDR
transfectants than for the pHaMDR transfectants. This
size difference as well as the difference in the colony
number at high concentrations of colchicine suggests
higher expression of ~DRl under control of the HaMSV
promoter than under control of the B-actin promoter in KB
cells. However, expression of MDR1 under control of the
truncated B-actin promoter (tBAP-MDR) is comparable or
higher than the expression of MDR1 under control of the
full-length B-actin promoter.
To confirm that the B=actin-MDRl expression
vectors function in mouse cells, these vectors were also
- 12 -
introduced in~o NIH 3T3 cells, and the colony-fo~ming
ability of parental NIH 3T3 cells and transfectants at 60
ng/ml colchicine (which is 3-5 fold as high as the LD50
of colchicine for the parental cells) measured. In this
e~periment (data not shown), a similar relative colony-
forming ability in the presence of colchicine was
achieved for NIH 3T3 transfectants with either the full-
length or truncated actin promoter construction, there-
fore, it was concluded that the truncated B-actin pro-
moter is an adequate promoter to express the MDRl cDNA in
transgenic mice.
Production of mice carryinq the BAP-MDRl
The 4.7 kb fragment obtained from an XhoI
digest of BAP-MDR (Figure lA) was microinjected into
B6SJL (Fl) fertilized eggs to generate MDRl transgenic
mice. The mice were screened by 50uthern blot analysis
of tail genomic DNA which was digested with EcoRI. Blots
were hybridized with the 5A probe (Figure 2A). Figure 2B
shows a Southern blot analysis of tail DNA of some mice,
showing the expected unique 3.1 kb internal fragment from
the MDRl cDNA, as well as the mouse endogenous fragments
which could be detected under hybridization conditions of
low stringency (Lanes 1-5) but not under high stringency
conditions (Lanes 6-10). For each blot analysis, the
full-length 4.7 kb injected fragment was diluted, mixed
with a negative mouse genomic DNA, digested with EcoRI
and applied on the gel as an internal control for com-
plete EcoRI digestion of the genomic DNAs. This sample
was also used as a standard to indicate the size as well
as to estimate the MDRl copy number in each transgenic
mouse (Lanes 2, 7). An EcoRI digest of human KB-3-1
genomic DN~ was also used to confirm the estimated MDRl
copy numher (Lanes 1, 6). The DNA samples from the MDRl
positive mice were also analyzed by slot blot analysis to
confirm the MDRl copy number in each transgenic mouse
(Figure 3A).
In ths study, five founder mice containing
2~
- 13 -
integrated human MDRl sequences were generated (Figure
3): Mouse 39 (female) had a low copy number (1-3
copies); mouse 132 (female) contained 100-200 copies of
MDRl; mouse 168 (male) carried around 50 copies of inte-
grated DNA; mouse 93 (male, with a high copy number)
showed an additional MDRl fragment in a Southern analysis
(which probably representPd rearranged DNA sequences or
junction fragments with host DNA) died soon after wean-
ing; mouse 104 (female, around 10 copies) killed her
first litter and died during her second pregnancy.
Transmission of MDR1 to progeny
Each of the remaining three transgenic founder
mice (M39, M132, and M168) was mated with a normal mouse
and tail genomic DNA samples from their progeny were
analyzed by Southern blot hybridization, as well as by
slot blot hybridization, for the presence of MDRl
sequence. Two of the founder mice (M39 and M132) trans-
mitted the integrated MDR1 sequences through the germ
line to about 50% of the progeny, as expected. No major
variations in the number or structure of the acquired DNA
sequences could be detected in Fl and F2 generations
(Figure 4, and data not shown). The DNA analyses of the
inheritance of the human MDRl cDNA in the third founder
(M168) revealed that out of the 32 first generation off-
spring, only 3 carried the introduced MD~l gene. There-
fore, it was concluded that this third founder mouse was
probably a mosaic.
Expression studies in MDR1 transqenic mice
The founder mouse M39 (low copy number) and its
positive progeny were mated with normal mice to generate
a MDRl heterozygous mouse line, termed line MDR-39. This
line was chosen for RNA expression studies. Total RNA
was prepared from 18 different tissues (including brain,
liver, kidney, spleen, heart, lung, stomach, small
intestine, colon, skin, tail, bone, bone-marrow, skeletal
muscle, ovary, uterus, oviduct, and testes) of 7 F2
transgenic mice (for pedigree see Fiqure 5) as well as of
- 14 -
negative sibling mice. These RNA samples were
analyzed by slot blot hybridization with MDR1 probes
(Figures 6 and 7). All blots were cross hybridized with
a s-actin probe as a standard control for accurate RMA
loading and filter transfer (Figure 6, and data not
shown). In each experiment, total RNA from multidrug-
resistant ~B cell lines expressing elevated levels of
MDR1 mRNA were included so that the unknown samples could
be directly compared with samples of known MDRl RNA con-
tent and known multidrug r~sistance. Relative to drug-
sensitive KB-3 1 t the multidrug-resistant subline KB-8-5
has a 40-fold increase in MDR1 mRNA and KB-V-1 (Vbl) has
a greater than 500-fold increase. These experiments,
summarized in Figure 6, reveal that the human MDR1 RNA is
expressed mainly in bone-marrow and spleen. Lower
expression was also detected in skeletal muscle and ovary
(Figure 7B~, but not in testes (data not shown). The
same blots were cross-hybridized with three different
contiguous MDRl probes (10, 5A, and P W II #6) which
together cover around 85% of the MDR1 cDNA (Figure 7A).
Except for skeletal muscle which hybridized significantly
more to the 5' probe (probe 10) than to the others, all
positive tissues showed similar results with all three
probes ~Figure 7B). These results indicate that in all
the tissues which express human MDR1 RNA, except for
skeletal muscle, the whole MDR1 mRNA was expressed.
Noxthern blot analysis (data not shown) showed an unde-
fined message size ranging from 4.5 (the expected full-
length MDRl message) up to around 11 kb. These results
indicate that the endogenous polyadenylation signal at
the 3' end of the M 1 cDNA worked weakly in the trans-
genic mice.
Out of seven MDR-39 mice tested, six showed
significant expression in bone marrow, with a somewhat
lower expression in spleen. However, one mouse showed
expression in spleen but not in bone marrow. One male
mouse showed some expression in kidney and liver in
~n~
addition to skeletal muscle as well as a high signal in
bone marrow and spleen. Although some expression was
detected in ovary but no~ in uterus and oviduct of
transgenic females, no expression was detected in testes.
Expression of P-qlycoprotein detected by immunofluores-
cence
Immunofluorescence with a mouse monoclonal
antibody directed against the human P-glycoprotein (MRK-
16) showed surface expression of human P-glycoprotein in
many different bone marrow cells in two MDR1 positive
mice, when compared with bone marrow derived ~rom a con-
trol sibling mouse (Figure 8). This could be clearly
seen in Figure 8 despite the presence oi background mouse
plasma IgG detected in the smear using rhodamine-
conjugated anti-mouse IgG.
In summary, the results presented herein clear-
ly demonstrate successful generation of transgenic mice
carrying and expressing the human MDR1 cDNA under the
control of a heterologous promoter. As shown herein
above, the strategy was to construct an expression vector
carrying the MDRl cDNA sequence 3' to a chicken B-actin
promoter. The B-actin promoter per se seems to be a
constitutive promoter and is only down-regulated by a
region 3' to the promoter sequences and by a small region
which is located 5' to the polyadenylation signal of the
B-actin gene itself. Neither of these inhibitory regions
were contained in the constructions of the present inven-
tion. A comparative study of DNA-mediated transfer of
expression vectors containing a cloned ~acterial chloram-
phenicol acetyltransferase (CAT) gene into a wide range
of cell types has shown that the B-actin promoter activ-
ity is equal to or stronger than the SV40 early promoter
(Gunning et al, 1987, Proc. Natl. Acad. Sci. USA 84:4831-
4835). Since B-actin is abundantly expressed in all
cells in culture, it was reasoned that a transgene carry-
ing MDRl sequences ~mder control of B-actin promoter may
express at a high level in a wide range of cell ~ypes,
- 16 ~
and ~herefore may confer drug resistance. To test this
assumption, first the ability of th~ B-actin MDRl expres-
sion v~ctor (pHG1) to confer a druy resistance phenotype
in cell culture was studied and indeed significant
acquired drug-resistance was found.
It has been shown that the presence of pro
caryotic vector sequences is highly inhibitory to appro-
priate expression of certain genes in transgenic mice.
Moreover, in mice carrying a transgene adjacent to proN
caryotic vector sequences, aberrations such as local
instability at the site of integration and insertional
mutagenesis are observed. In order to eliminate vector
sequences in th0 pronuclear injected DNA in accordance
with the present invention, an ~hoI fragment of pHGl was
isolated. This strategy also deleted 60 bp in the 5'
region of the B-actin promoter. To test the expression
ability of this truncated B-actin promoter, an identical
plasmid to pHGl, but lacking the upstream 60 bp of the B-
actin promoter (pHG2), was constructed and transfected
into human and mouse cells in culture. These studies
established that the truncated B-actin promoter activity
is equal to or greater than the full-length promoter in
cultured cells. Hence, in accordance with the present
invention, the XhoI B-actin MDRl fragment was found ade-
quate to introduce the human MDRl cDN~ into mice.
From the generated MDR1 founder mice and their
progeny lines, one line (MDR-39) was chosen for expres-
sion studies. Restriction fragment pattern analyses of
genomic DNAs from this founder and over 100 descendant
mice (Figure 5, data not shown) indicate that 1-3 copies
of the injected fragment were integrated into the mouse
DNA. Also, neither internal rearrangement, deletion nor
duplication could be detected. Pedigree analyses indi-
cated that the transgene is: (a) transmitted through the
germ line; (b) integrated :into a single chromosome, since
the transgene is stably inherited by about 50% of the
progeny of matings of heterozygous and normal mice; and
~3~
(c) inherited in an autosomal ~ashion and is neither x-
or y- linked, since male-to-male and female-to-female
transmission of the tran~gene occurred.
It is known that the insertion of foreign D~A
sequence into ~he cellular genome can cause muta-tional
changes by disrupting the ~unction of endog2nous genes or
of control elements. Most insertional mutations in
transgenic mice are recessive, but in most of the matings
between heterozygous mice their embryonic lethal pheno-
type is demonstrated by significan~ly reduced litter size
and by the inabilit~ to produce homozygous mice. Since
homozygous matings of MDR-39's progeny revealed normal
litter size, and phenotypically normal homozygotes could
be generated (data not shown), it was concluded that no
insertional mutation of endogenous housekeeping or con-
trol genes occurred during the integration of the trans~
gene in this mouse line.
RNA studies of the MDR-39 transgenic mice
showed that the transgene is expressed mainl~ in hemo-
poietic -tissues such as bone marrow and spleen. Lower
expression is de~ected in skelet~l muscle and ovary.
However, no phenotypic and morphologic changes nor func-
tional or behavioral disturbances could be detected in
the transgenic mice studied.
Primer extension studies of RNA extracted from
tissues expressing the transgene ~ith a human specific 35
base synthetic oligonucleotide (which does not cross-
hybridize to mouse mdr sequences at conditions o~ high
stringency) (Ueda et al, 19~7, J. Biol. Chem. 262:505-
508), resulted in a single extension product of around
192 bases, as expected if the MDR1 cDNA is expressed
under the control of the B-actin promoter. These results
suggest a single major transcrip-tion initiation site of
the transgene under control of the B-actin promoter,
rather than under a mouse endogenous promoter adjacent to
the integration site.
Quantitative measurements of RNA expression
'~5)0~
- 18 -
levels in bone marrow of MDR-39 transgenic mice sho~ed
relatively high expression of MDRl. The expression level
is comparable to, or in some mice up to 3-fold greater
than expression levels of _ 1 mRNA in KB-8-5 drug
resistant cells. These levels (which are 40~ to 120-fold
higher than the basal expression level in drug sensitive
cells) are at least as high as MDRl RNA levels in multi-
drug resistant human tumors.
The present invention now makes it possible for
MDRl transgenic mice which express the human MDR1 gene in
bone marrow to be resistant to doses of chemotherapy
which would be toxic to the bone marrow of the normal
mice. For example, an intraperitoneai dose of the chemo-
therapeutic agent daunomycin produces bone marrow
suppression and death 2-3 weeks after treatmen~ in the
majority of the mice so treated. In contrast, the trans-
genic mice of the presen~ invention are resistant to that
dosage of a chemotherapeu~ic agent which would be lethal
to normal mice. Hence, by establishing tumors in MDRl
transgenic mice (either by transplantation or by intro-
duction of genetic alterations which predispose the mice
~o such tumors, or by other suitable methodology~, the
tolerance limits of potent chemotherapeutic preparations,
containing either a single or a combination of drugs, can
be determined.
Transgenic mice of the present invention also
allow genetic manipulations to improve suitability of the
MDR-3g and other MDR1 transgene mice for various pur-
poses. These manipulations include the matings of MDR1
mice to produce mice homozygous at the MD~l locus and to
produce fully inbred strains of mice for tumor xenograEt
studies. Furthermore, by crossing MDR1 mice with nude
immunodeficient mice, animals are obtained that accept
human tumors while yet being multidrug resistant. This
allows the testing of tolerable limits and efficacy of
anti-tumor or anti-cancer agents.
An additional utility of the cloned MDRl is as
-- 19 --
a dominant selectable marker to introduce linked genes
into the bone marrow and other tis~ues of humans and
animals. The MDRl mice allow the determination of the
dosages of drugs (such as daunomycin, vincristine,
vinblastine, VP-16, VM-26, actinomycin D, adriamycin and
the like) needed to select cells or tissues expres~ing
the human MDRl gene and introduced into recipient host.
This is accomplished by using MDR1 mice to determine the
tolerable dosages of these and other chemotherapeutic
agents for bone marrow and other tissueæ expressing MDR1
gene. Of course, such studies are needed prior to devel-
opment o~ effective gen0 therapy regimens using the MDR1
gene as a selectable marker.
The MDR1 transgenic mice also allow the study
of the role of multidrug transporter encoded by this
gene. Expression of the MDR1 gene at relatively high
levels in ti~sues in which this gene is not normally
expressed, allows the determination of the physiological
effects of the transporter on various tissues. Further-
more, the MDR1 transgenic animals can serve as donors for
various cells and tissues expressing the human MDR1
gene. These cells can be established in culture or can
be used as the sources of multidrug resistant tissues in
animal transplant experiments.
It is understood that the examples and embodi-
ments described herein are for illustrative purposes only
and that various modifications in light thereof will be
suggested to persons skilled in the art and are to be
included within the spirit and purview of this applica-
tion and scope of the appended claims.
- 20 -
Table 1. Frequency of drug-resistant colonies
Colonies per dish
Plasmid 5Colchicine (ng/ml) __
pHaMDR 620 130 25
pBAP-MDR 680 32
ptBAP-MDR 890 46 7
No DNA 0 0 0
KB-3-1 (1 x 10-6 cells per 10-cm dish) were trans-
fected with 10 ug of DNA; 48 hr after transfection, cells
w~re split into 4 dishes and cultured in the presence of
colchicine at the indicated concentration. On day 12,
cells were stained and colonies counted.
