Production of Transgenic Live-Bearing Fish andCrustaceans with Replication-Defective PantropicRetroviral Vectors
Aliye Sarmasik,* C.Z. Chun, In-Kwon Jang,† J.K. Lu,‡ and Thomas T. Chen§
Biotechnology Center and Department of Molecular and Cell Biology, University of Connecticut, 184 Auditorium Road,
U-149, Storrs, CT 06269, USA
Abstract: Transgenic fish have been routinely produced by microinjecting or electroporating foreign DNA into
one-cell stage embryos or unfertilized eggs. While both techniques are effective in producing transgenic fish
species from which unfertilized or newly fertilized eggs can be easily obtained, these techniques are not
applicable to live-bearing fish and many crustacean species where unfertilized or newly fertilized eggs are not
readily available. In this paper, we describe a new method of introducing foreign DNA into the live-bearing fish,
Poeciliposis lucida, and crayfish, Procambarus clarkii, by directly transforming the immature ovary or testis of
these animals with replication-defective pantropic retroviral vectors carrying a reporter gene (neoR). A signifi-
cant fraction of the progeny derived from these treated animals contains the neoR reporter gene, determined by
a PCR-based assay. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50%
of the resulting progeny carried the transgene, suggesting that the F1 animals are germline transgenic. Inte-
gration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated
with transgene constructs. The expression of reporter genes was detected by reverse transcription (RT) PCR
assay. These results showed that foreign genes could be reproducibly transferred into live-bearing fish and
crustaceans by directly transforming the immature gonads with replication-defective pantropic retroviral vec-
tors.
Key words: replication-defective pantropic retroviral vectors, live-bearers, crustaceans; gene transfer; transgenic
animal.
INTRODUCTION
Organisms into which foreign DNA (transgene) has been
artificially introduced and stably integrated in host genomes
are called transgenics (reviewed by Gordon, 1989; Jaenisch,
1990). Since 1985, a wide range of transgenic fish species
have been produced by microinjecting or electroporating
foreign DNA into newly fertilized or unfertilized eggs (re-
Received January 31, 2001; accepted March 30, 2001.
*Current address: Department of Aquatic Organism Rearing, College of Aquaculture,
Canakkale Onsekiz Mart University, Canakkale 17100, Turkey.
†Current address: Department of Aquaculture, West Sea Fisheries Research Institute,
National Fisheries Research and Development Institute, Inchon 400-201, South Korea.
‡Current address: Department of Aquaculture, National Taiwan Ocean University, Kee-
lung, Taiwan.
§Corresponding author: telephone 860-486-5011; fax 860-486-5005; e-mail
[email protected]
Mar. Biotechnol. 3, S177–S184, 2001DOI: 10.1007/s10126-001-0040-3
© 2001 Springer-Verlag New York Inc.
viewed by Chen and Powers, 1990; Fletcher and Davis,
1991; Hackett, 1993; Chen et al., 1995, 1998). Several im-
portant steps are routinely taken to produce a desired trans-
genic fish (Chen et al., 1998): (i) selection of an appropriate
species; (ii) preparation of a transgene construct; (iii) in-
troduction of the transgene into unfertilized or newly fer-
tilized fish eggs; and (iv) selection and characterization of
the resulting transgenic fish. However, low rates of trans-
gene integration and germ-line mosaicism in P1 transgenic
individuals are some of the limitations of these gene transfer
methods (Chen et al., 1995, 1998). Furthermore, since the
foreign DNA needs to be microinjected or electroporated
into newly fertilized or unfertilized eggs, these methods are
only applicable to species whose fertilized or unfertilized
eggs are readily obtainable.
Poecilid fishes, such as guppies, platyfish, swordtails, or
topminnows, have been widely used as model experimental
animals in cancer research and in monitoring chemical pol-
lutants in aquatic ecosystems (Morizot et al., 1990). These
fish are live-bearers, which consequently, limits the possi-
bility of conducting gene transfer studies in them by the
conventional methods of microinjection and electropora-
tion. In many crustacean species important to aquaculture,
fertilization is initiated by transferring spermatotheca from
males to females, and by the time that embryos are released
from females, they are at advanced developmental stages.
Consequently, foreign genes cannot be transferred into
their unfertilized or newly fertilized eggs by microinjection
or electroporation. Alternative methods for producing
transgenic animals in crustaceans as well as in live-bearing
fish would be highly desirable.
Recently Burns and colleagues (Burns et al., 1993; Yee
et al., 1994) developed a series of new gene transfer vectors,
broad-host-range (pantropic) replicative-defective retrovi-
ral vectors containing the long terminal repeat (LTR) se-
quence of Moloney murine leukemia virus (MoMLV) and
transgenes (e.g., neoR or b-gal) packaged in a viral envelop
with the G-protein of vesicular stomatitis virus (VSV) vec-
tors. These vectors were effective in infecting cell lines of
fish, newt, Xenopus, and mosquito (Burns et al., 1993, 1994;
Miyanohara et al., 1992; Matsubara et al., 1996) and in
newly fertilized finfish and shellfish eggs such as those of the
medaka, zebrafish, and surf clam (Burns et al., 1993; Lu et
al., 1996, 1997), because these vectors contain the G-protein
from the vesicular stomatitis virus (VSV) that can bind to
phospholipids of the cell membrane. Stable transgenic
medaka and surf clams have been produced by electropor-
ating these vectors into newly fertilized embryos (Lu et al.,
1996, 1997). We believed that these pantropic defective ret-
roviral vectors could also infect immature gonads in situ
and result in the prodcution of transgenic individuals by
crossing transformed animals with their untransformed
counterparts. To test this hypothesis, we introduced pan-
tropic defective retroviral vectors carrying a neoR reporter
gene into immature gonads of Poeciliopsis lucida and Pro-
cambarus clarkii. Transgenic F1 individuals of both species
were produced by crossing reproductively active, treated
animals with untreated animals, and detailed characteriza-
tion of transgenic animals revealed transgene integration
into the genomes of F1 transgenic individuals. Furthermore,
transgene transmission and transgene expression also were
detected in F1 and F2 progeny. This approach offers new
possibilities of introducing desirable genes into live-bearing
fish and crustaceans for basic research and biotechnological
applications.
MATERIALS AND METHODS
Retroviral Vector Constructs and Treatmentof Animals
The replication-defective pantropic retroviralvector,
LSRNL-(VSV-G), in which the MoMLV LTR drives the
expression of the hepatitis B surface antigen (HbsAg) and
the RSV LTR drives the expression of the neomycin phos-
photransferase gene (neoR), was prepared and titered as pre-
viously described (Burns et al., 1993; Yee et al., 1994). The
production of Geo4.8 was also described previously (Burns
et al., 1994).
Immature Poeciliposis lucida (36 males and 21 females)
at the age of 2 months (20–25 mm in length) or immature
crayfish (20 each of males and females) at the age of 3
months were injected with 3–4 µl of a LSRNL or Geo4.8
pantropic retroviral vector (1.1 × 106 CFU/ml) in the vi-
cinity of gonads. The injected individuals were reared in
aquarium under a photoperiod of 14 hours light/10 hours
dark at 25°C until reproductive maturation (at 3 months for
P. lucida and 41⁄2 months for the crayfish), and mated to
untreated animals in single-pair mating.
Identification of Transgenic Fish
The presence of transgene in presumptive transgenic indi-
viduals was determined by PCR amplification of the trans-
gene sequence and confirmation of the transgene identity
by southern blot hybridization. Genomic DNA samples
S178 Aliye Sarmasik et al.
were isolated from small pieces of fin tissues collected from
presumptive transgenic individuals and untreated controls
following the standard phenol–chloroform method de-
scribed by Sambrook et al. (1989). PCR amplification was
conducted using 1.0 µg of genomic DNA as a template and
neoR gene specific oligomers as amplification primers under
the following amplification conditions: 30 seconds at 94°C
for denaturation, 30 seconds at 60°C for annealing and 30
seconds at 72°C for synthesis for 35 cycles. The neoR gene
specific oligonucleotides (forward primer 58-GCATTGC-
ATCAGCCATGA-38 and reverse primer 58-GATGGATTGCACGCAG-
GTTC-38) are used as PCR amplification primers.
Following PCR, the products were electrophoresed on
a 1.2% agarose gel, transferred to nylon membranes and
hybridized to [32P]-dCTP-labeled neoR transgene (1.0 × 106
cpm/ml) prepared by PCR amplification of the pLSRNL
plasmid DNA with neoR gene specific oligomers as ampli-
fication primers under the same conditions described
above. Hybridization was carried out at 65°C for 16 hours
in a solution containing 6 × SSC (1× solution: 0.15 M NaCl,
0.015 M Na citrate), 5× Denhardt’s solution (1× solution:
0.02% polyvinylpyrrolidine, 0.02% BSA, 0.02% Ficoll),
0.5% SDS, 0.1% yeast RNA, and 0.05% sodium pyrophos-
phate. The blots were washed once in 1× wash solution [1×
SSC, 0.1% sodium dodecyl sulfate (SDS), and 0.05% so-
dium pyrophosphate], once in 0.5× wash solution and once
in 0.1× wash solution at 58°C, and exposed to x-ray films at
−80°C.
Detection of Transgene Expression
The expression of the neoR gene in the transgenic animals
was determined by PCR following reverse transcription
(RT). Total RNA was isolated from transgenic individuals
by the guanidium thiocyanate phenol chloroform extrac-
tion (Chomczynski and Sacchi, 1987). Single-stranded
cDNA (sscDNA) was synthesized by reverse transcription
using 2.5 µg of total RNA as a template and oligo-(dT)-
adaptor as a primer. The reaction mixture (20 µl final vol-
ume) contained 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3
mM MgCl2, 0.5 mM dNTP, 10 U RNase inhibitor (Perkin
Elmer) and 100 U of Superscript II (BRL) and was incu-
bated at 42°C for 50 minutes, then heat inactivated for 10
minutes at 70°C. The transgene cDNA sequence was am-
plified by 30 cycles of PCR with oligomers specific to the
neoR as amplification primers. The PCR products were elec-
trophoresed on a 1.0% agarose gel, transferred to nylon
membranes, and hybridized to 5 × 106 cpm/ml of [32P]
dCTP-labeled neoR. Conditions of hybridization and blot
washing were as described in the previous section.
Integration of Transgene
To detect the integration of transgene in F1 transgenics, a
PCR integration assay was employed following conditions
described by Lin et al. (1994) and modified by Burns et al.
(1996). Genomic DNA samples were isolated from whole
fish as described (Sambrook et al., 1989), digested to
completion with restriction endonuclease KpnI and re-
solved on a 1% agarose gel. DNA fragments between 1 and
3 kb that contain viral LTR and the flanking host genomic
DNA were eluted from the gel. The recovered DNA frag-
ments were ligated with T4 DNA ligase and the LTR se-
quence was amplified by PCR using a set of oligomers spe-
cific to the LTR sequence (58-TTTGAAAGACCCCACCCG-38 and
58-AATGAAAGACCCCCGTCG-38) as amplification primers. The
PCR products were resolved on a 1.2% agarose gel, trans-
ferred to nylon membrane and hybridized to a [32P]-dCTP-
labeled probe (5 × 106 cpm/ml) prepared by PCR amplifi-
cation of the LTR sequence from the plasmid pLSRNL,
using primer set described above. The conditions of hybrid-
ization, blot washing and autoradiography were the same as
described in the previous section.
RESULTS AND DISCUSSION
Studies conducted by Burns et al. (1993, 1994) and those in
our laboratory (Lu et al., 1996, 1997) showed that replica-
tion-defective pantropic retroviral vectors are effective in
transferring foreign DNA into many established cell lines
and embryos of medaka and surf clams, because these vec-
tors contain the G-protein of the vesicular stomatitis virus
(VSV). These results suggested that replication-defective
pantropic retroviral vectors might also serve as effective
vehicles to transfer foreign genes into liver-bearing fish or
crustaceans via transforming the primordial germ cells. Fol-
lowing the strategy outlined in Figure 1, a pantropic repli-
cation-defective retroviral vector (LSRNL or Geo4.8) carry-
ing neoR reporter gene was delivered by intraperitonal in-
jection near the gonads of immature Poeciliposis lucida and
crayfish. About 50% of both female and male crayfish and
Poeciliopsis lucida survived from the treatment. After repro-
ductive maturation, the treated animals were mated to un-
treated counterparts and the progeny collected for PCR
analysis for the presence of neoR transgene following the
Gene Transfer in Live-Bearing Fish and Crustaceans S179
Figure 1. Strategy for producing
transgenic live-bearing fish and
crustaceans by directly transforming the
immature gonads with
replicative-defective pantropic retroviral
vectors.
Figure 2. Identification of presumptive
transgenic animals by PCR analysis.
Transgene sequence was amplified from
genomic DNA samples of putative
transgenic animals by PCR, and the PCR
products were resolved on 1.0% agarose
gels, transferred to nylon membranes,
and hybridized to 32P-labeled neoR
transgene probe. (a) Strategy of PCR
analysis. (b) Ethidium bromide stain gel
pattern of PCR products from putative
transgenic crayfish (Procambaqrus clarkii).
Lane M, molecular size markers; lanes
1–4, DNA from putative transgenic
individuals; lane 5, pLSRNL plasmid
DNA. (c) Southern blot hybridization of
PCR products from putative transgenic
Poeciliposis. Lane 1, no DNA template;
lanes 2–7, genomic DNA samples from
putative F1 individuals.
S180 Aliye Sarmasik et al.
Table 1. Inheritance of neoR Transgene in F1 Generation
Sex of P1
F1 analyzed
(N)
PCR positive
(N)
Transgenics
(%)
Poeciliposis lucida
Male 19 4 21
Male 14 8 57
Male 34 23 68
Male 21 6 28
Male 24 14 58
Female 24 1 4
Procambarus clarkii
Male 12 4 33
Female 12 8 67
Table 2. Inheritance of neoR Transgene in F2 Generation
Family
F2
analyzed
(N)
PCR positive
(N)
Transgenics
(%)
Poeciliposis lucida
F1a × nontransgenic 12 6 50
F1b × nontransgenic 20 9 45
F1c × nontransgenic 35 19 54
F1d × nontransgenic 20 14 70
F1e × nontransgenic 20 12 60
Procambarus clarkii
F1a × nontransgenic 15 9 60
F1b × nontransgenic 15 8 57
F1c × nontransgenic 15 7 47
F1a–F1e: male or female fish selected from different F1 families.
Figure 3. Detection of transgene
integration by PCR amplification. (a)
Schematic presentation of detection of
transgene integration. Genomic DNA from
F1 transgenic and nontransgenic
individuals were digested to completion
with KpnI and size fractionated on 1%
agarose gels. DNA fragments of 1–3 kb
were recovered from the gel, ligated, and
the LTR sequence associated with the 1 to
3-kb DNA fragments amplified by PCR.
PCR products were resolved on 1.2%
agarose gels, transferred to nylon
membranes, and hybridized to 32P-labeled
LTR probe. (b) Detection of transgene
integration in transgenic and nontransgenic
Poeciliopsis lucida. Lane 1, DNA from
pLSRNL plasmid after KpnI digestion, size
fractionation, and ligation; lane 2, DNA
from nontransgenic animal; lanes 3–5,
genomic DNA samples of different
transgenic animals. (c) Detection of
transgene integration in transgenic and
nontransgenic crayfish. Lane 1 genomic
DNA of nontransgenic animal; lanes 2–4,
DNA samples of different transgenic
animals.
Gene Transfer in Live-Bearing Fish and Crustaceans S181
strategy described in Figure 2a. About 83% (15/18 animals)
of the surviving males and 9% (1/11 animals) of females of
Poeciliposis lucida carry the proviral vector in their gonads.
Table 1 shows patterns of proviral transmission to the F1
generation in Poeciliposis lucida and crayfish. The neoR
transgene was carried by several to 70% of the F1 progeny
derived from different treated parents (P1 animals). These
results suggest that in both species of animals only a fraction
of the germline in each P1 animal was transformed by pro-
viral vectors. When the F1 animals derived from different P1
transgenic lines were mated with nontransgenic controls,
about 50% of the resulting F2 progeny carried the neoR
transgene (Table 2), following a Mendelian segregation pat-
tern. These results suggest the proviral vectors are present
throughout the entire germline of each F1 transgenic line.
To confirm the integration of proviral vectors into the
host genome of F1 transgenics, genomic DNA samples iso-
lated from different F1 transgenic individuals of Poeciliposis
and the crayfish were digested with KpnI to completion, the
resulting DNA fragments resolved on agarose gels, and
DNA fragments of 1–3 kb were recovered from the gel.
Following ligation, PCR amplification was conducted to de-
termine the presence of the LTR sequence in the 1 to 3 kb
DNA fragments. As shown in Figure 3, a predicted 588-bp
LTR sequence was amplified from DNA samples of several
F1 transgenic animals, suggesting that the proviral vector
LTR was associated with the host genome.
Since the integration of retroviral sequence into the
host genome requires cell division, we selected primordial
germ cells as the target of infection by the retroviral vectors
because of their capacity for cell division. The timing of
initiating gonad infection was very critical because the
number of dividing primordial germ cells in the gonad de-
creases as the animal approaches reproductive maturation
in any reproductive cycle. Under standard rearing condi-
tions in our laboratory, Poeciliposis lucida reach reproduc-
tive maturation at three months. To ensure successful in-
tegration of retroviral vectors into the germline, we infected
Figure 4. Detection of neoR transgene
expression in transgenic Poeciliopsis and
crayfish. The neoR mRNA in F1
transgenic animals was detected by
RT-PCR assay. Two microliters of 1/10
diluted cDNA products were used as
templates for PCR assay. The products
were resolved on 1% agarose gels. (a)
Detection of neoR mRNA in transgenic
crayfish. RT-PCR products were resolved
on 1% agarose gels and the DNA bands
visualized by staining with ethedium
bromide. Lanes 1 and 2, RNA from
transgenic crayfish I; lanes 3 and 4, RNA
transgenic crayfish II; lane 5, RNA from
nontransgenic crayfish; and lane 6,
Geo 4.8 plasmid DNA. Lanes 2 and 4,
PCR without prior reverse transcription.
(b) Detection of neoR mRNA in
transgenic Poeciliposis. The RT-PCR
products were transferred to a nylon
membrane and hybridized to 32P-labeled
neoR probe. Lanes 1 and 4, RNA from
transgenic fish I; lanes 2 and 5, RNA
from transgenic fish II; lanes 3 and 6,
RNA from transgenic animal III and lane
7, DNA of LSRNL-(VSV-G). Lanes 1–3,
PCR without prior reverse transcription.
S182 Aliye Sarmasik et al.
them at an age not older than 2 months. Crayfish reach to
sexual maturity at the age of 41⁄2 months under standard
rearing conditions, and therefore, the gonads were infected
with pantropic retroviral vectors at age 3 months. Since we
produced a significant number of transgenic Poeciliposis and
crayfish in our studies, the time that we choose to initiate
the infection process in both animal species must be the
appropriate point.
The expression of the neoR transgene in transgenic Poe-
ciliposis and crayfish was determined from several F1 indi-
viduals by RT-PCR analysis, and the results are presented in
Figure 4. A 347-bp RT-PCR product showing the expres-
sion of neoR transgene was detected in many F1 transgenic
animals but not in nontransgenic controls. These results are
in good agreement with those reported by Burns et al.
(1994), Lu et al. (1996, 1997) and Matsubara et al. (1996).
Pantropic replication-defective retroviral vectors previ-
ously were used to produce transgenic fish (Lin et al., 1994;
Lu et al., 1997) and shellfish (Lu et al., 1996). In those
studies, however, the gene transfer vectors were transferred
into newly fertilized embryos by microinjection or electro-
poration. In many aquatic animal species such as live-
bearing fish and crustaceans, newly fertilized or unfertilized
eggs cannot be readily obtained, and thus, the prerequisite
of obtaining newly fertilized or unfertilized eggs will limit
their use as model organisms for conducting gene transfer
studies. In our studies, pantropic replication-defective ret-
roviral vectors directly transformed, in situ, the immature
gonads of Poeciliposis and crayfish, thus bypassing the need
for microinjecting or electroporating proviral vectors into
newly fertilized or unfertilized eggs to produce transgenics.
To our knowledge, this is the first report of successful gene
transfer in a live-bearing fish and a crustacean species.
In summary, we report successful germline transfor-
mation and expression of transgenes in a live-bearing fish
(Poeciliposis lucida) and crayfish (Procambarus clarkii) by
treating their immature gonads with pantropic replication-
defective retroviral vectors. Our data demonstrate retroviral
infection and stable integration of the provirus in the germ-
lines of Poeciliposis and crayfish for three generations. Fur-
thermore, expression of the neoR transgene in F1 progeny of
both transgenic animal species demonstrates that the RSV
promoter can mediate foreign-gene expression in live-
bearing fish and crustaceans. Therefore, we believe that
pantropic retroviral vectors will allow the transfer of supe-
rior genetic traits, such as fast somatic growth or disease
resistance, into economically important crustacean species
for commercial aquaculture. Furthermore, these gene trans-
fer vectors will facilitate the generation of transgenic model
live-bearing fish with reporter genes for studies in environ-
mental toxicology and cancer research.
ACKNOWLEDGMENT
This research was supported by grants from NSF (IBN-
9723529 and IBN-0078067), USDA (#CONS-9803641 and
CONTR #58-1930-0-009) and Connecticut Sea Grant Col-
lege (R/A 18) to T.T.C.
REFERENCES
Burns, J.C., Friedmann T., Driever, W., Burrascano, M., and Yee,
J.K. (1993). Vesicular stomatitis virus G glycoprotein pseudotyped
retroviral vectors: concentration to very high titer and efficient
gene transfer into mammalian and nonmammalian cells. Proc Natl
Acad Sci USA 90:8033–8037.
Burns, J.C., Matsubara, T., Lozinski, G., Yee, J.K., Friedmann, T.,
Washabaugh, C.H., and Tsonis, P. (1994). Pantropic retroviral
vector-mediated gene transfer, integration, and expression in cul-
tured newt limb cells. Dev Biol 165:285–289.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of
RNA isolation by acid guanidinium thiocyanate-phenol-
chloroform extraction. Anal Biochem 162:156–159.
Chen, T.T., and Powers, D.A. (1990). Transgenic fish. Trends Bio-
technol 8:209–215.
Chen, T.T., Lu, J.-K., and Kight, K. (1995). Transgenic fish. In:
Molecular Biology and Biotechnology, Meyers, R.A. (ed.). VCH
Publishers, Inc., 910–914.
Chen, T.T., Lu, J.-K., and Fahs, R., II. (1998). Transgenic fish
technology and its application in fish production. In: Agricultural
Biotechnology, Altman, A. (ed.). New York: Marcel Dekker, Inc.,
527–547.
Fletcher, G.L., and Davis, P.L. (1991). Transgenic fish for aqua-
culture. In: Genetic Engineering, Vol. 13, Setlow, J.K. (ed.). New
York: Plenum Press, 331–370.
Gordon, J.W. (1989). Transgenic animals. Int Rev Cytol 155:171–
229.
Hackett, P.B. (1993). The molecular biology of transgenic fish. In:
Biochemistry and Molecular Biology of Fish, Vol. 2, Hochachka, P.,
and Mommsen, T. (eds.). Amsterdam: Elsevier Science Publishers,
207–240.
Gene Transfer in Live-Bearing Fish and Crustaceans S183
Jaenisch, R. (1990). Transgenic animals. Science 240:1468–1477.
Lin, S., Gaiano, N., Culp, P., Burns, J.C., Friedmann, T., Yee, J.K.,
and Hopkins, N. (1994). Integration and germ-line transmission
of a pseudotyped retroviral vector in zebrafish. Science 265:666–
668.
Lu, J.K., Chen, T.T., Allen, S.K., Matsubara, T., and Burns, J.C.
(1996). Production of transgenic dwarf surfclams, Mulina lateralis,
with pantropic retroviral vectors. Proc Natl Acad Sci USA 93:3482–
3486.
Lu, J.K., Burns, J.C., and Chen, T.T. (1997). Pantropic retroviral
vector integration, expression, and germline transmission in
medaka (Oryzias latipes). Mol Mar Biol Biotechnol 6:289–295.
Matsubara, T., Beeman, R.W., Shike, H., Besansky, N.J., Mukaba-
yire, O., Higgs, S., James, A., and Burns, J.C. (1996). Pantropic
retroviral vectors integrate and express in cells of the malaria mos-
quito, Anopheles gambiae. Proc Natl Acad Sci USA 94:6181–6189.
Miyanohara, A., Elam, R.L., Witztum, J.L., and Friedmann, T.
(1992). Efficient in vivo transduction of the neonatal mouse liver
with pseudotyped retroviral vectors. New Biol 4:261–267.
Morizot, D.C., Schultz, R.J., and Wells, R.S. (1990). Assignment of
six enzyme loci to multipoint linkage groups in fishes of the genus
Poeciliopsis (Poeciliidae): designation of linkage groups III-V. Bio-
chem Genet 28:83–95.
Sambrook, J., Fritsch, E., and Maniatis, T. (1989). Molecular Clon-
ing: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Yee, J.K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J.K., and
Friedmann, T. (1994). A general method for the generation
of high-titer, pantropic retroviral vectors: highly efficient infec-
tion of primary hepatocytes. Proc Natl Acad Sci USA 91:9564–
9568.
S184 Aliye Sarmasik et al.
