Note: Descriptions are shown in the official language in which they were submitted.
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MICROINJECTION PROCEDURE FOR GENE TRANSFER IN FISH
FIELD OF THE INVENTION
This invention relates to the transforming of the
genome of a fish egg to inherent new genetic traits.
BACKGROUND OF THE INVENTION
There are many situations where if the genetic
make-up of the fish species could be altered, vast
improvements would be realized in the realizable food
value of fish stock. The transfer of various genetic
traits from one fish to another to enhance growth,
disease resistance, environmental resistance rate, meat
quality and the like could be realized.
For example, it has been well established that
certain marine fish can survive icy water, that is water
at temperatures of 0 C or below. The types of fish
which survive such water temperature do so by
synthesizing anti-freeze proteins or glycoproteins and
secreting them in their body fluids. These anti-freeze
compounds in the body fluids act non-colligatively to
depress the freezing temperature of the body fluids and
thus the freezing temperature of the fish. However,
there are many other types of fish, such as the salmonid
species which do not produce anti-freeze polypeptides.
These anti-freeze proteins have been studied
extensively in the Winter Flounder which is known to
produce anti-freeze polypeptides during the winter.
These peptides are a-helices, rich in alanine, with
molecular weights ranging from 3,300 to 4,500.
It has been determined that the anti-freeze
polypeptide genes from the Winter Flounder consist of a
gene family with approximately 30 to 40 members.
Approximately two-thirds of this family are arrayed in
direct tandem repeats with the remainder linked, but
irregularly spaced. The tandemly linked anti-freeze
polypeptide genes code for the major anti-freeze
polypeptide components found in Winter Flounder plasma.
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It is very difficult, however, to transfer genes of
one fish to another, because of the very sensitive
nature of the fish egg and the inability to ensure that
the injection leads to incorporation of the transferred
DNA into the fish genome. This is particularly
difficult in those types of fish eggs which tend to be
opaque thus rendering location of the fish egg nucleus
difficult.
SUMMARY OF THE INVENTION
According to one aspect of the invention, a method
is provided for transferring into a fish egg DNA of at
least one gene foreign to the fish egg so as to
integrate the foreign DNA stably into the fish egg
genome. The method comprises:
inserting a hollow needle into the fish egg,
through the micropyle thereof, and injecting
therethrough and into the fish egg a selected volume of
a solution containing a plurality of copies of the
foreign DNA.
By another aspect of the invention, there is
provided a transgenic salmonid species of fish which
actively expresses a gene coding for an anti-freeze
protein (AFP) when the fish is exposed to icy water.
By yet another aspect of the invention, there is
provided fertilized eggs treated in accordance with the
method of this invention wherein the foreign DNA is at
least one gene encoding for anti-freeze polypeptides.
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are
discussed with respect to the drawings wherein:
Figures la and b are schematic representations of
the micropyle injection site of a representative fish
egg;
Figure 2 is a restriction map of the integrated
linearized plasmid 2A-7 which includes the entire Winter
Flounder anti-freeze gene;
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Figure 3 is a genomic Southern Blot of salmon DNA
as probed with nick-translated anti-freeze polypeptide
gene containing Sst 1 fragment from 2A-7 plasmid; and
Figure 4 is a genomic Southern Blot of salmon DNA
as probed with nick-translated plasmid pUC-9.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It is understood that a variety of genomic traits
may be transferred to fish in accordance with this
invention. As aforementioned, such traits include
disease resistance, environmental condition resistance,
growth-promoting hormones such as growth hormone and
growth hormone releasing factor genes and many other
beneficial genes, meat qualities and the like. To
exemplify the manner in which this invention may be
applied for transferring various genetic traits to fish,
the invention will be discussed with reference to the
transfer of anti-freeze properties to the salmonid
species of fish. The gene or genes encoding for anti-
freeze properties may be isolated from a variety of fish
which survive in icy waters. A particularly suitable
source for this trait is the Winter Flounder.
The inventors have discovered that injection of the
desired number of copies of the foreign DNA into the egg
has to be conducted through the micropyle of the
fertilized egg to achieve stable integration of the
foreign DNA into the fish egg genome. With reference to
Figures la and b, the micropyle site is representatively
shown. In Figure la, the micropyle site is designated M
which is in the region of the blastodisc BL. The yolk
is encased in the chorion designated CH. As with all
types of fish eggs, there is the pervitelline space PV
as well as the vitelline membrane VM. The yolk Y is
shown as occupying a substantial portion of the space
within the egg chorion CH. As shown in Figure lb, the
micropyle is enlarged with a schematic representation
of fertilizing spermatozoa FS entering through the
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micropyle. In this representation, the micropyle
includes the micropilar canal MC which opens within the
egg containing the cytoplasm CY adjacent the metaphase
of second maturation division MII. In addition, a
first polar body PBI is shown adjacent the opening of
the micropyle to the cytoplasm as well as a cortical
alveoli CA.
In accordance with this invention, injection of
the foreign DNA into the fish eggs has resulted in a
survival rate to hatching of approximately 80%, which
is comparable to the survival rate of normal uninjected
eggs. Hence the injection technique does not appear to
affect the survival rate. Furthermore, by injection
through the micropyle site, the inventors have
discovered that there is stable integration of the
foreign DNA into the fish genome. With such stable
integration, there is an expression of the anti-freeze
gene which produces the anti-freeze polypeptides to
enable the fish transformed with the foreign DNA to
survive sub-zero temperatures.
With reference to Figure 2, the restriction map
of the linearized plasmid 2A-7 is shown. This plasmid
is readily produced by the technique disclosed in Scott
et al., (1985), "Anti-Freeze Protein Genes are Tandemly
Linked and Clustered in the Genome of the Winter
Flounder", Proc. Natl. Acad. Sci. U.S.A. 82:2613-2617.
This genomic subclone contains a 7.8-kb BamHI fragment
of Winter Flounder DNA cloned into the BamHI site of
plasmid pUC 9. This fragment was derived from a X-
charon 30 Flounder genomic DNA library and represents
one of the tandem repeats that each contain one copy of
the 1-kb anti-freeze polypeptide gene. The 2A-7
subclone can be prepared by banding in CsCl/ethidium
bromide and linearized by digestion with Eco RI. The
digested DNA can be extracted with phenol/chloroform
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13453
recovered by ethanol precipitation and stored in a
Tris/EDTA solution.
A sufficient number of copies of the DNA of Figure
2 are prepared by suitable recombinant DNA techniques in
5 producing multiple copies of the plasmid. In accordance
with a preferred embodiment of this invention, it is
desired to use in the range of 106 copies for each
injection. The copies are carried in a suitable
solution which is injected through the micropyle of the
fertilized egg by use of an appropriate needle. It has
been found that hollow glass needles having an outside
diameter in the range of 3 to 5 m and driven by a
micro-manipulator are particularly suitable. To provide
for a 2 to 3 nL injection of the DNA copies in solution,
a device is employed which provides short bursts in the
range of 100 to 1,000 microseconds of nitrogen at 200
kPa. The timing of these bursts of nitrogen can be
controlled by a device such as a Grass 544 Stimulator
(Grass Instruments, Quincy, MA). By use of this device
and this type of needle construction, the small volumes
of injection into the egg are readily achieved and
needles of this diameter, which can be readily inserted
through the micropyle of the egg, can be withdrawn
without damaging the egg. By injection of the copies of
the DNA through the micropyle, it has been found that
the foreign DNA is inserted in a region of the egg which
provides for incorporation of one or more of the copies
of the foreign DNA into the fish genome. It has been
the inventors' experience that this cannot be
accomplished by microinjection into other locations of
the fish egg.
The following Examples exemplify various preferred
aspects of the invention which are not to be
interpreted as limiting the scope of the appended
claims.
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Preparations and Materials
Sources of Fish EaQs
Atlantic salmon adults (305 kg) were captured from
the Exploits and Colinet river systems (Newfoundland)
and transported live to the Marine Sciences Research
Laboratory, Memorial University, Newfoundland, 2 to 3
weeks prior to spawning. At the laboratory, male and
female salmon were maintained at seasonally ambient
photoperiod in 2 x 2 x 0.5 m deep aquaria supplied with
flowing freshwater and air.
Eggs and sperm were stripped from salmon which had
been anaesthetized in a dilute solution of t-amyl
alcohol. The eggs were stored up to 3 days in plastic
containers at 4 C until use. Sperm were stored in 20-mL
vials and checked for motility under a compound
microscope just prior to fertilizing the eggs.
Eggs were fertilized up to 2 hours prior to
microinjection and rinsed with several changes of ice-
cold (4 C) salmon Ringer. The composition of the salmon
Ringer solution was as follows:
NaCl 6.5 gm
KC1 0.25 gm
NaHCO3 0.2 gm
CaC12-2H20 0.4 gm
make up to 1 liter with distilled water.
When this procedure was used, the eggs were
fertilized but not activated. Activation occurred when
the eggs were placed in a hypotonic solution [Ginsburg,
A.S. (1963) "Sperm-egg Association and its Relationship
to the Activation of the Eggs in Salmon Fishes", J.
Embryol. Exp. Morpho. 11:13-33].
Preparation of DNA
The DNA used for microinjection was the genomic
subclone 2A-7 which contained a 7.8-kb BamHI fragment of
Winter Flounder DNA cloned into the BamHI site of
plasmid pUC 9 [Scott, G.W. et al, (1985) "Antifreeze
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Protein Genes are Tandemly Linked and Clustered in the
Genome of the Winter Flounder" Proc. Natl. Acad. Sci.
USA 82:2613-2617] (see Figure 2). This fragment was
derived from a A charon 30 flounder genomic DNA library
and represents one of the tandem repeats that each
contain one copy of the 1-kb anti-freeze polypeptide
gene. The 2A-7 subclone was prepared by banding in
CsCl/ethidium bromide and was linearized by digestion
with Eco RI. The digested DNA was extracted with
phenol/chloroform, recovered by ethanol precipitation
and stored frozen in 10 mM Tris-HC1 (pH 7.4)/ 1mM EDTA.
Example 1 - Microiniection
The injection apparatus consisted of a hollow glass
needle 3-5 pm (outside diameter) driven by a micro-
manipulator. The volume of DNA solution injection (2 to
3 nL) was regulated using short bursts (100 to 1000 ms)
of N2 at 200 kPa. The timing of the bursts was
controlled using a Grass 544 Stimulator (Grass
Instruments, Quincy, MA). Eggs were injected with - x
106 copies of linearized clone 2A-7 through the
micropyle within 3 hours of fertilization and incubated
in freshwater at 8 C. The micropyle, which is located
in close proximity to the egg nuclear area, was
visualized using a dissecting microscope at 20 times
magnification (Ginsburg, A.S. (1986) "Fertilization in
Fishes and the Problem of Polyspermy", Academy of
Sciences of the USSR, Institute of Development Biology,
Translated from Russian by Z. Blake and edited by B.
Golek, (Israel Program for Scientific Translations,
Jerusalem 1972, Keter Press Binding:Wiener Binding Ltd.,
Jerusalem p 366; Pringle, H. (1987) "Superfish", Eauinox
32:68-77 and Riehl, R. (1980) "Micropyle of Some
Salmonins and Coregonins", Environ. Biol. Fishes, 5:59-
66].
Example 2 - Incorporation of Foreign DNA
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Thirty fingerlings (1 to 2 g each) collected 8
months after injection were frozen in liquid N2 and
stored at -70 C for analysis. Genomic DNA was prepared
from individual fingerlings by the method of Blin and
Stafford (1976) "Isolation of High Molecular-Weight
DNA", Nucleic Acids Res, 3:2303-2308, as modified by
Scott et al, (supra). To test for the incorporation of
the flounder anti-freeze polypeptide gene, salmon
genomic DNAs (15 pg) were digested with a threefold
excess of restriction enzymes for 3 hours at 37 C,
electrophoresed in 0.7% agarose gels, and blotted onto
either nitrocellulose or Biotrace [Southern, E.M. (1975)
"Detection of Specific Sequences among DNA Fragments
Separated by Gel Electrophoreses", J. Mol. Biol. 98:503-
517]. Southern blots were probed with a gel-purified,
nick-translated 2.7-kb Sst 1 fragment from subclone 2A-7
that completely encompasses the anti-freeze polypeptide
gene. Blots on Biotrace' were stripped of probe after
autoradiography and were rehybridized with nick-
translated pUC-9.
Of the 1800 Atlantic salmon eggs injected with DNA,
approximately 80% survived to hatching. This survival
rate is essentially the same as that of uninjected eggs.
For preliminary screening, genomic DNAs were digested
with Sst 1 and BamHI and Southern blotted. Two DNAs (26
and 36) from the 30 individual fingerlings tested showed
hybridization signals with the anti-freeze polypeptide
gene probe in an initial screening (not shown). These
two DNAs were retested along with a negative control
(45, one which showed no hybridization signals) and
again showed hybridization to the probe (Figure 3). The
Sst 1 digests (S)of 26 and 36 both resulted in a band of
'c hybridization at -2.7 kb which corresponded to one of
the major Sst--i-fragments that hybridized in Winter
Flounder (wt) genomic DNA. This is in effect identical
to the Sst 1 fragment present in subclone 2A-7 (Figure
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2) and referred to as the "b" polymorphism in Scott et
al (supra) which was used as the hybridization probe in
Figure 3. This hybridization signal is not seen in the
control (45), even though the ethidium bromide stained
pattern of mass bands indicated that this DNA has been
digested to the same extent as 26 and 36 (not shown).
Digestion of 26 and 36 with BamHI (B) produced a main
band of hybridization at 7.8 kb (Figure 3), consistent
with predictions from the map in Figure 1 and several
minor bands. These two samples differed in their
hybridization pattern after Hind III digestion. In 26
the major band of hybridization appeared at 13.5 kb,
while in 36 it was 9.4 kb. As in the BamHI lanes, there
were several minor bands of hybridization. Finally, in
the undigested samples, the only hybridization signal
coincided with the band of high molecular weight genomic
DNA.
To study the fate of the plasmid vector which
remained attached to linearized 2A-7, the blot shown in
Figure 3 was stripped of hybridization probe and
reprobed with labelled pUC 9(Figure 4). There is a
significant amount of hybridization to the undigested
DNA in the control lanes of 26 and 36 but no bands of
hybridization below this high molecular weight material
and only background hybridization to the undigested
flounder DNA or 45 DNA. Several bands of hybridization
were present in all three enzyme digests of sampled 26
and 36 which were absent from the single digest of 45.
The length of these bands generated with each enzyme
were different from one DNA to the other as they would
be if they represented independent integrations into the
genome.
The results of Figures 3 and 4 demonstrate the
hallmarks of a stably integrated copy of the Winter
Flounder AFP gene derived from linearized 2A-7. In all
transgenic fish, Sst 1 and BamHI should liberate the
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gene in 2.7- and 7.8-kb restriction fragments,
respectively. Hind III should give a gene-containing
fragment of at least 7.8 kb, but with a varied upper
length. The latter prediction holds because there is
5 only a single Hind III site in 2A-7 in the multiple
cloning site of the pUC 9 portion. If a single copy of
2A-7 is integrated, then the second Hind III site must
come from the flanking salmon genomic DNA. When the
same digests are probed with the 2.7-kb of pUC 9
10 plasmid, they show an entirely different pattern of
digestion, with minimum lengths for the hybridization
signal of 2.7 kb in both the BamHI and Hind III digests
and a minimum of -4.7 kb in the Sst 1 digest.
The most likely explanation for the minor bands in
the BamHI and Hind III lanes of 26 and 36 is that they
represent cleavage products of 2A-7 that have been
independently incorporated into the salmon genome. In
this situation, one would expect to see fewer of these
accessory bands in the Sst 1 lanes than in the other two
digests, simple because there is less chance of the
breaks in 2A-7 occurring within the central 2.7 kb Sst 1
fragment than within the 7.8-kb BamHI fragment. When
the overall hybridization signal present in 26 and 36 is
compared with the signal derived from the 30 to 40 genes
in the Sst 1 digested Winter Flounder standard (Figure
3), it is clear that more than one copy of the AFP gene
had been incorporated into the transgenic salmon.
Although preferred embodiments of the invention
have been described herein in detail, it will be
understood by those skilled in the art that variations
may be made thereto without departing from the spirit of
the invention or the scope of the appended claims.
