WO2000075288A1

WO2000075288A1 - Transgenic decapods, decapod cell lines and methods of producing same

Info

Publication number: WO2000075288A1
Application number: PCT/US2000/015083
Authority: WO
Inventors: Jane C. Burns; Hiroko Shike
Original assignee: The Regents Of The University Of California
Priority date: 1999-06-04
Filing date: 2000-06-01
Publication date: 2000-12-14
Also published as: AU5312200A

Abstract

Transgenic decapods and methods of producing transgenic decapods use pseudotyped retroviral vectors containing the vesicular stomatitis virus (VSV) G protein. The VSV-G protein confers broad host range on the retroviral vectors.

Description

TRANSGEMC DECAPODS, DECAPOD CELL LINES AND METHODS OF PRODUCING SAME

Field of the Invention The present invention relates to transgemc decapods, particularly shrimp, and to methods for producing transgemc decapods using pantropic retroviral vectors.

Description of the Related Art Commercial shrimp aquaculture is currently plagued by infectious disease problems associated with intensive cultivation. In particular, two viruses, white spot and yellow head, are threats to the commercial shrimp enterprise in the Americas. No method currently exists to combat these infectious diseases. In particular, penaeid shrimp are economically important species that are distributed throughout Asia, Australia, and the Western Hemisphere. Wild and farmed populations are under considerable pressure from indigenous and imported viruses that threaten the stability of these populations (Lightner, CDC Handbook of Maπculture , 1:393-486, 1993). Understanding the cellular biology, immune response, and pathogenesis of viral infections in these species will be critical in designing strategies to ensure their survival.

Pantropic retroviral vectors have the ability to infect a broad range of species (Burns et al., Proc. Natl. Acad. Sci. U.S.A. 90:8033-8037, 1993; Matsubara et al., Proc. Natl. Acad. Sci. USA 93:6181-6185, 1996; Lu et al., Proc. Natl. Acad. Sci. U.S.A. 93:3482-3486, 1996; U. S. Patent No. 5,969,211, hereby incorporated by reference), and provide a tool for the genetic manipulation of non mammalian species. These viral vectors cannot replicate by themselves. After entering the cell, the virus particle is dismantled and no further infectious particles can be created.

Foreign genes can be carried by the vector into the cell and can be stably integrated into the host cell genome. In this way, all daughter cells receive this genetic information. If the information is introduced into germline cells (eggs or sperm), the genetic material will be passed on to future generations.

Retroviral vectors based on the munne and avian leukemia viruses have become standard tools for the introduction and expression of foreign genes in mammalian cells, both in vitro and in vivo. To create a retroviral vector, the coding sequences for the structural gene (gag), the reverse transcnptase gene [pol), and the envelope protein gene (env) are removed and replaced with heterologous DNA under the control of the retroviral promoter (long terminal repeat, LTR) or an internal promoter. These replication- incompetent vectors are produced in packaging cell lines that express the retroviral proteins needed for the assembly of an infectious particle. The vector particles can be recovered from the culture supernatant and used to infect target cells.

Vectors based on the oioney munne leukemia virus ( oMLV) have been pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) substituted for the retroviral envelope protein (Burns et al., supra.).

The VSV-G protein binds to phospholipid moieties in the cell membrane, thus circumventing the need for a specific protein receptor on the target cell surface (Mastromaπno et al., J. Gen. Virol. 68:2359-2369, 1987). These pseudotyped retroviral vectors, therefore, have an expanded host cell range (pantropic) and can be concentrated to titers > 10⁹ cfu/ml by ultracentπfugation (Burns et al., supra.; Yee et al., Meth. Cell Biol. 43:99 112, 1994). Pantropic vectors can attach, uncoat, reverse transcribe, and integrate into the genome of a wide variety of species including insects, fish, and marine invertebrates (Matsubara et al., supra.; Jordan et al., Insect Mol. Biol. 7:215 222, 1998 ; Lin et al. Science 265:666-668, 1994; Lu et al., Molec. Marine Biol. Biotechnol. 6:289 295, 1997; Lu et al., Proc. Natl. Acad. Sci. U.S.A. 93:3482-3486, 1996).

Classical agricultural practices include hybridization, in which entire genome sets are exchanged between different species, and selective breeding, in which animals carrying desired traits are inbred to establish a stable breeding line. Primary culture of different shrimp cell types has been achieved and primary cell cultures have been maintained for weeks to months, particularly in the presence of growth factors added to the tissue culture medium (Chen et al.. Fish Pathol. 21:161 -166, 1986, Luedeman et al., Aquaculture 101:205-211, 1992, Nadaia et al., In Vitro

Cell Dev. Biol. 29A:620-622, 1993, Tapay et al., Proc. Soc. Exptl. Biol. Med. 209:73 78, 1995, Hsu et al., Aquaculture 136:43 55, 1995; Toullec et al., J. Crustacean Biol 16:643-649, 1996). Stable expression of foreign genes, however, has not been convincingly demonstrated. These approaches have significant disadvantages and rely heavily on chance to obtain the desired goal. Thus, there is a need for a reliable method for producing transgemc decapods and decapod cell lines which have desirable genetic traits. The present invention addresses this need.

Summary of the Invention One embodiment of the present invention is a method for introducing exogenous ONA into a decapod, comprising: infecting a decapod embryo with a retroviral pseudotype comprising the exogenous DNA, the retroviral pseudotype comprising: retroviral long terminal repeats (LTRs); RNA corresponding to a promoter within said LTRs, RNA corresponding to the exogenous DNA, wherein the exogenous DNA is operably linked to the promoter; and VSV-G protein eπcapsidatiπg the RNA. Preferably, the pseudotype is prepared by: inserting the exogenous DNA into a first plasmid comprising the retroviral LTRs and the promoter such that the exogenous DNA is operably linked to the promoter; and forming the retroviral pseudotype by expressing the plasmid in a cell comprising a VSV G gene and retroviral gag and pol genes. In one aspect of this preferred embodiment, the LTRs are MoMLV LTRs. Advantageously, the plasmid comprises a selectable marker. Preferably, the decapod is a shrimp. In another aspect of this preferred embodiment, the forming step comprises: transfecting a packaging cell line with the first plasmid, wherein the packaging cell line assembles vector particles containing an RNA copy of the exogenous DNA; infecting a producer cell line with the retroviral particles, the producer cell line comprising the retroviral gag and pol genes; and transfecting the vector particle-containing producer cell line with a second plasmid containing a promoter operably linked to the VSV G protein, whereby pseudotyped retroviral vectors containing the VSV-G protein are secreted by the producer cell line. Preferably, the packaging cell line is PA317. Advantageously, the producer cell line is 293 cells. In another aspect of this preferred embodiment, the second plasmid is pHCMV G. Preferably, the promoter of said second plasmid is cytomegalovirus promoter. The method may additionally comprise dechonoπating the embryo of the decapod prior to infecting. The present invention also provides a method for introducing exogenous DNA into a decapod cell line, comprising: infecting a decapod cell with a retroviral pseudotype comprising the exogenous DNA, the retroviral pseudotype comprising: retroviral long terminal repeats (LTRs); RNA corresponding to a promoter within said LTRs, RNA corresponding to the exogenous DNA, wherein the exogenous DNA is operably linked to the promoter; and VSV G protein encapsidating the RNA. Preferably, the pseudotype is prepared by: inserting the exogenous DNA into a first plasmid comprising the retroviral LTRs and the promoter such that the exogenous DNA is operably linked to the promoter; and forming the retroviral pseudotype by expressing the plasmid in a cell comprising a VSV-G gene and retroviral gag and pol genes. Preferably, the decapod is a shrimp.

Another embodiment of the invention is a decapod expressing exogenous DNA. Preferably, the decapod is a shrimp.

The present invention also provides a decapod cell line expressing exogenous DNA. Preferably, the decapod is a shrimp.

Brief Description of the Drawings Figure 1 A shows the organization of the RNA genome of pantropic retroviral vectors. LTR - Moloney munne leukemia virus long terminal repeat; luc- firefly lucif erase; RSV- Rous sarcoma virus LTR; neo- neomycm phosphotransferase; hsp70- Drosophila heat shock protein 70 promoter; IE-1 - AcMNPV baculovirus (Autographa californica multicapsid nuclear polγhedrosis virus) immediate early- 1 promoter; Z- £ coll -galactosidase.

Figure 1 B shows a schematic comparison of infectious and "bald" vector particles lacking an envelope glycoprotein. The vector particles contain identical retroviral proteins but lack the genes that encode for these proteins.

Figure 2 is a graph showing luciferase expression in shrimp Oka organ primary cell cultures infected with the retroviral vectors LLRNL, LNGIAI LucL, LNhsp70LucL and mock infected (-).

Figure 3 is a graph confirming the results of the experiment shown in Figure 2. LLRNL infection was repeated on the Oka organ primary cultures in triplicate. In addition, cells were exposed to LLRNL "bald" virus that lacks an envelope protein and is non-infectious. The experiment was performed on Oka organs isolated from two individual shrimp. Luciferase activity is expressed as light umts/mg total cellular protein. Luciferase activity from

LLRNL infected cells expressed as mean of triplicate infections + 1 S.D.

Figure 4 shows a comparison of four promoters driving luciferase expression. Results of three separate experiments: cells from Oka organ were pooled from two shrimp (Expt. 1), from three shrimp (Expt. 2), or cultured from a single shrimp (Expt. 3). "Bald" LLRNL- non-infectious control; LNIEIucL = infectious vector with luciferase gene expressed from immediate-early promoter of insect baculovirus Autographa californica Multicapsid Nuclear Polyhedrosis Virus (AcMNPV). LNhsp70LucL= infectious vector with luciferase gene expressed from Drosophila heat shock 70 promoter; LLRNL - infectious vector with luciferase gene expressed from Drosophila heat-shock 70 promoter. Figure 5 shows the effect of timing and duration of exposure to the retroviral vector, LLRNL, on luciferase expression. Three independent experiments were performed with LLRNL applied to the cultures at 3 different time points after preparation of the Oka organ cultures. In Experiments 1 and 2, the cultured cells were derived from the Oka organ of a single shrimp/experiment. Experiment 3 was performed on pooled Oka organ cells from 2 shrimps. Figure 6 shows the effect of vector dose on luciferase expression. Oka organ cells were pooled from three shrimp and exposed to different concentrations of LLRNL. Bar shows mean lights umts/mg protein of triplicate assays + 1 S.D.

Detailed Description of the Preferred Embodiments Pantropic vectors that infect a wide variety of species were used to deliver and express reporter genes in cultured cells from the blue shrimp, Penaeus stylirostπs. This is the first report of foreign gene expression in a penaeid shrimp and of pantropic retroviral vector infection and gene expression in primary cultured cells from a marine arthropod. The direct comparison of heterologous promoters for the expression of foreign genes demonstrated that two different retroviral LTRs mediate easily detectable levels of gene expression. The present invention provides a method for transferring exogenous DNA sequences into decapods and decapod cell lines using pseudotyped retroviral vectors containing the genome of a retrovirus and the envelope glycoprotein of vesicular stomatitis virus (VSV) (i.e. VSV G protein) which confers broad host range. In addition, the invention provides transgemc decapods and decapod cell lines. The term "decapod" refers to any of an order (Decapoda) of crustaceans including shrimp, lobsters, crayfish and crabs, having five pairs of thoracic appendages, one or more or which are modified into pincers, with stalked eyes, and with the head and thorax fused into a cephalothorax and covered by a carapace. In a preferred embodiment, transgemc shrimp are produced using pantropic retroviral vectors Successful transductioπ and expression of different reporter genes was demonstrated in primary cultured cells derived from the Oka organ and from the ovaries of mature shrimp. The retroviral LTR mediated readily detectable levels of reporter gene expression. The following experiments describe the successful introduction of exogenous DNA sequences into primary cultured shrimp cells and shrimp eggs, the integration of these sequences into the genome of the resulting larval stages, and the expression of these exogenous DNA sequences. The exogenous DNA will also be incorporated into the germ line (eggs and sperm) once the shrimp reach sexual maturity. Once these larva are raised to sexual maturity and mated, the resulting progeny (FI generation) will also contain and express the exogenous DNA. The presence of the exogenous DNA can be determined using well known methods such as polymerase chain reaction (PCR) or Southern hybridization. Expression of the exogenous DNA can be determined using standard assays for the activity of the gene product encoded by the exogenous DNA sequence.

As used herein, the term "pantropic" indicates a retroviral pseudotype in which the envelope glycoprotein is VSV-G, and the term "provirus" indicates the integrated DNA form of the virus. The substitution of the VSV-G glycoprotein for the amphotropic retroviral envelope protein confers upon the resultant pseudotyped vector particles a broadened host range and the ability to be concentrated to high titer by ultraceπtrifugation. These pseudotyped pantropic retroviral vectors can infect many mammalian and non mammalian species. For example, U. S. Patent No. 5,969,21 1 , the enfre contents of which are hereby incorporated by reference, describes the insertion of exogenous DNA into the genome of mollusks. Because the vector particles are replication deficient, they can be safely used following guidelines for recombiπaπt DNA techniques (BSL2). The organization of the RNA genome of pantropic retroviral vectors is shown in Figure 1 A. Figure 1 B shown schematic comparison of infectious and "bald" vector particles lacking an envelope glycoprotein. The vector particles contain identical retroviral proteins but lack the genes that encode for these proteins.

The ability to manipulate the genome of commercially important species of shrimp will allow the creation of unique broodstock carrying desirable genetic traits, such as disease resistance and growth acceleration. Thus, the ability to selectively introduce a single gene into the genome of a target species of shrimp without otherwise disturbing the genome of the animal would be beneficial. There are currently no bona fide examples of stable expression of foreign genes in decapods. Decapod cell lines expressing various foreign genes can be exploited for the study of shrimp cellular biology and regulation of gene expression The present invention demonstrates for the first time that exogenous genes can be introduced into shrimp cells and shrimp, and that these exogenous genes can be expressed. The genetic engineering of shrimp will allow insertion of disease resistance genes and other desirable traits, such as enhanced growth. As discussed below, pantropic retroviral vectors can infect, uncoat, reverse transcribe and stably integrate into the shrimp genome.

The shrimp system described herein is meant to be illustrative of the invention as a model system for studying decapod genetics. Accordingly, the disclosure relating to this species and gene transfer technique is not intended to limit the present invention. Thus, the introduction of exogenous nucleic acid sequences into other decapods such as lobsters and crabs, and into other decapod cell lines, is also within the scope of the invention.

In a preferred embodiment, exogenous DNA is introduced by incubation of pantropic retroviral vector comprising exogenous DNA with shrimp embryos in which the chonon has been damaged or removed by physical or chemical treatment, such as by treatment with the tryptophan terminator 3 ammo 1,2,4 tnazole (ATA). Other methods of DNA introduction contemplated for use in the invention include microinjectioπ and electroporation. Low voltage eiectroporation transiently disrupts the vitelline membrane, allowing direct contact of the virus particles with the embryo surface, but does not compromise the integrity of the embryo.

Invertebrate defense genes which confer resistance to various pathogens may be transferred into the decapod genome. These defense genes include antimicrobial peptides such as the cecropiπs, magaimns, diptencins, defensiπs and attacins (Hoffman et al., Phylogenetic Perspectives in Immunity: The Insect Host Defense, R.G. Landers Co., Austin, TX, 1994; Cociancich et al., Parasito/ogy Today, 10:132 139, 1994). This will improve the resistance of mollusk populations to disease. Genes encoding growth regulating hormones will accelerate moilusk growth and result in larger animals. For example, Paynter et al. (Biol. Bull. 181:459-462, 1991 ) treated oysters with recombinant trout growth hormone. The growth hormone-treated oysters were significantly larger than control oysters. The introduction of additional gene copies of, for example, growth-accelerating hormones into various decapod species will accelerate growth of these organisms, resulting in larger decapods for harvesting and consumption. Because the exogenous growth-accelerating genes are under control of a constitutive promoter, growth will be greatly accelerated due to the inability of endogenous transcription control factors to down regulate expression of the growth hormone gene. In a preferred embodiment, the polycation polybrene can also be used to facilitate virus attachment to the embryo surface. The use of other polycations including, for example, protamme sulfate and poly L-lysine, is also within the scope of the invention.

The envelope protein of pantropic retroviral vectors interacts with phosphohpids in the plasma membrane to mediate virus attachment. The absence of a requirement for a specific protein receptor on the cell surface confers an extremely broad host cell range upon these vectors. In addition, the stability of the envelope protein allows concentration of vector particles to high titer by ultracentnfugation as described in U. S. Patent No. 5,512,421, the entire contents of which are hereby incorporated by reference. These retroviral vectors can accommodate up to about 10 kilobases of heterologous cDNA and promoter sequences, are easy and inexpensive to produce, and mediate stable insertion of the retroviral genome carrying transgenes into the host cell genome. No other system exists for the efficient and stable introduction of exogenous DNA into a decapod species.

Any pantropic retroviral vector is contemplated for use in the invention. Pantropic retroviral vectors include VSV G-pseudotyped vectors, such as LSRNL(VSV-G), LSPONL(VSV G), LZRNL(VSV-G), LLRNL(VSV-G), LNIEI LucL(VSV-G) and LNhsp70lucL(VSV-G). In these retroviral vectors, L- Moloney munne leukemia virus (MoMLV); S- hepatitis B virus surface antigen; R- Rous sarcoma virus LTR; N- neomycm phosphotransferase; hsp 70=- Drosophila heat shock protein 70; luc- luciferase; IE1 - baculovirus immediate early promoter, P0 - polio virus internal nbosomal entry site; and VSV-

G - vesicular stomatntis virus envelope protein. Piasmids pLSPONL and pLZRNL are described by Yee et al. {Proc. Natl. Acad. Sci. U.S.A. 91:9564 9568, 1994). Plasmid pLNhsp70lucL is described by Jordan et al. {Insect Mol. Biol. 7:215 222, 1998). Plasmid pLLRNL is described by Xu et al. {Virology 171:331-341, 1989) and is commercially available from Clontech (Palo Alto, CA) for research purposes only. Plasmid pLNIEUucL is described by Franco et al. (Insect Biochem. Mol. Biol. 28:819-825, 1998). All of these constructs contained the neomycm resistance gene (N) to allow selection in medium containing G418. The VSV G pseudotγpes corresponding to these constructs can be constructed as described in Example 2 of allowed Application Serial No. 08/844,530.

The use of other rhabdovirus envelope glycoproteins is also contemplated in the generation of pseudotyped pantropic retroviral vectors for use in the invention. MoMLV based retroviral vectors are particularly preferred. For production of retroviral vector particles, the nucleic acid of the vector particles of the present invention can be used to transfect a suitable packaging cell line. Typically, the suitable cell line expresses the gag and pol genes required for retroviral replication. In a preferred embodiment, the vector particle contains DNA encoding a drug resistance gene (e.g., neomycm resistance). Cells containing the vector particles are selected by incubation in the corresponding drug (e.g., G418). The resistant cells are expanded and traπsfected with a plasmid containing a gene which encodes an envelope glycoprotein which confers broad host range upon the pseudotyped retroviral vector. The vector particles are released into the supernatant from the transfected cells, titered using an appropriate cell line, and used to infect decapod embryos.

The gene encoding VSV-G can be incorporated within the nucleic acid of the vector particle. Alternatively, the gene for this envelope protein can be expressed from a third fragment of nucleic acid or from the genome of the producer cell. In a preferred embodiment, the nucleic acid within the vector particle is integrated into the cellular genome of the cell infected by the vector particle and the envelope gene is located on a different fragment of nucleic acid than the nucleic acid that is vector particle genome. Thus, in this preferred embodiment, the membrane- associated protein is not produced by the vector particle infected cells containing the integrated nucleic acid from the vector particle. The following examples are illustrative of various steps for carrying out the present invention. These examples are provided for illustration purposes only and are not intended to limit the invention.

Example 1

Production of pseudotyped retroviral vector particles An amphotropic packaging cell line such as PA317 (ATCC CRL 9078) which expresses retroviral gag, pol and env was transfected with the plasmid of interest (Miyanohara et al., New Biologist 4:261-267, 1992). After 48 hours, cell culture containing amphotropic vector particles was harvested and used to infect the human adenovirus 5- traπsformed embryonal kidney packaging cell line 293 (ATCC CRL 1573) containing the Moloney munne leukemia virus (MoMLV) gag and pol genes (Burns et al., Proc. Natl. Acad. Sci. U.S.A. 90:8033-8037, 1993). Cell clones containing the plasmid of interest were selected by cultunng in the presence of the antibiotic G418, and neomycin-resistant clones were expanded. The 293 producer cells assemble vector particles; however, these particles are nomnfectious because they lack a viral envelope protein.

The plasmid infected 293 cells were transfected with 20 μg pHCMV-G (ATCC 75497) which expresses VSV G from the human cγtomegalovirus promoter. The culture medium was replaced with fresh medium 8 hours after transfection and the pseudotyped virus was collected between 24 and 96 hours post-transfectioπ. The titer of the virus was determined on rat 208F fibroblasts.

Example 2

Collection of viable, fertilized shrimp eggs The collection of viable shrimp eggs and infection with retroviral vectors represented a tremendous technical challenge which required a great deal of experimentation. Due to the fragility of the eggs and spawning requirements, the collection and rearing conditions had to be determined in order to obtain viable eggs for infection. In addition, the concentration and nature of a dechoπonatiπg agent had to be determined. Mated female shrimp (P. stylirostπs) were transferred to a spawning tank, kept in darkness and monitored with a flashlight until the spawning moment. When the animals initiated swimming and spawning, they were gently captured by net, and placed into a one-liter beaker containing 400-700 ml of Pacific seawater from the same tank until they completed spawning as described

(Muralidharan et al., Biol. Bull. 174:181-185, 1988). During spawning, the animals were calm and numerous eggs were collected into the beaker. Almost all embryos collected by this procedure burst within 5-10 min. By light microscopic examination, embryo deformity was recognized, followed by the rupture of the vitelline envelope and cell membrane, and release of the granules inside the embryos. This phenomenon was repeated for eggs collected from three different females spawned over three different days.

As an alternate approach, embryos were collected without holding the female. While the female was swimming and spawning, the egg-dense part of seawater below this animal was quickly scooped by the same beaker.

More than 500 eggs collected into the beaker were observed to undergo normal development. The eggs of P. sty/irostris are about 200 μM in diameter at spawning and start to sink towards the bottom. The net with less than

180 μM holes was used for effective collection of the freshly spawned shrimp eggs.

Example 3

Infection of dechorionated shrimp eggs with retroviral vectors

In order to infect freshly spawned shrimp eggs, the chorion is damaged or removed to create channels through which retroviral particles can pass. Freshly spawned eggs were collected in a 2-liter beaker and the tryptophan terminator 3-amino-1,2,4-triazole (ATA, Sigma #A8056) was added immediately to a final concentration of

0.2 mM. This compound has been used to remove the hatching envelope of the penaeid shrimp, Sicyonia ingentis, at concentrations of 0.1-1 mM (Hertzler et al., J. World Aquaculture Soc. 24:1-5, 1993). Eggs were allowed to sink to the bottom, and supernatant sea water was discarded to concentrate the eggs. Two methods were tried to infect embryos (about 60 min old and in 2-4 cell stage).

In method 1, the retroviral vector LNhsp70lucL (MoMLV IJ E.co/i heat shock protein 70-luciferase-MoMLV

LTR/ (8 x 10⁵ cfu total) and polγbrene (final cone. 2 μg/ml) were added to sea water containing about 30 embryos.

After 1 hour, 200 ml more sea water was added to dilute the ATA and polybrene. Embryos were kept at room temperature. In method 2, embryos were placed on two tissue culture inserts (20-30 embryos/insert), and 1 ml of sea water containing retroviral vector (1 x 10⁵ cfu total/insert) and polybrene (2 μg/ml) was flowed-through. After 1 hour, the inserts were immersed in 200 ml fresh sea water and kept at room temperature. The next day, both groups of hatched embryos (nauplii; first free swimming larval stage) were transferred into 500 ml bottles and oxygenated.

Nauplii (2 days old) were pelleted by ceπtrifugation and DNA was extracted for polymerase chain reaction (PCR) analysis.

About 10-15 nauplii hatched from both experimental groups (methods 1 and 2) and DNA was extracted for

PCR analysis using primers for the LTR (Jordan et al., supra.). Both batches of retrovirus-exposed nauplii gave a positive signal by nested LTR PCR. Negative control DNA from uπinfected nauplii was negative. The DNA extracted from all three groups of nauplii were positive by PCR with positive control actin primers, thus verifying the adequate quantity and quality of the extracted DNA. The lower limit of detection of the LTR PCR assay is about 2,000 infected cells/organism. This result shows that ATA-treated eggs at the 2-64 cell stage can be infected with retroviral vector. Thus, infection, reverse transcription and integration of the provirus into the shrimp genome occurred.

Example 4 Infection of shrimp embryos

Freshly spawned eggs were collected in a 2-lιter beaker and ATA was added immediately to a final concentration of 0.2 mM to dechononate the eggs. Eggs were allowed to sink to the bottom over 10 15 mm, and half of the supernatant sea water was discarded, leaving a volume of 1 liter sea water plus embryos. Embryos were aliquoted into five disposable plastic cups (200 ml/cup). After embryos settled to the bottom of the cup, supernatant sea water was drained out by using a plastic tube (internal diameter ^■ 4 mm) as a sump pump. The final concentration of the embryos was about 30/10 ml of sea water in a cup with a bottom diameter of 6 cm. Forty-five minutes after spawning, polybrene (final cone. 2 μg/ml) was added to all the groups. Three 10 ml aiiquots were incubated for one hour with three different retroviral vectors: (LNIELucL, LLRNL and LNhspLucL), one aliquot with non-infections retroviral vector (retrovirus with no envelope, called bald virus), and the last aliquot with no vector. After one hour, more sea water (about 150 ml) was added to dilute the ATA and polybrene. The hatched nauplii were raised to zoea, the second free swimming larval stage, and analyzed by PCR 72 hours after the initial infection for the detection of provirus.

Microscopic examination after vector incubation revealed embryos at 4 8 cell stage with irregularity and tiny holes in the choπon, confirming the expected effect of ATA. Because room temperature was 22°C and could be very cold during the night, a water bath was set up and all five cups were transferred to the water bath. In addition, aeration with tubing was set up in each cup. The next day, only 10 nauplii had hatched, all from the group of embryos incubated with LNIELucL. No nauplii hatched from the four other aiiquots. The water bath was 30°C, and the aeration tube had accidentally come off the fifth aliquot (no treatment group) (Table 1 )

Table 1

^'Titer is based on the infectious colony forming units on 208F rat fibroblasts. Non infection "bald" virus cannot be titered, but was prepared in the same way as the infectious virus with one round of ultraceπtπfugation. The presence of a similar number of viral particles was confirmed with PCR amplification of partially transcribed LTR genome.

Embryos were collected and treated with ATA as previously described. Various volumes of sea water containing embryos were aliquoted into four 200 ml tissue culture flasks (75 cm²) in the vertical position, resulting in approximately 40 embryos in 200 ml sea water, 30 embryos in 150 ml, 20 embryos in 100 ml, and 10 embryos in 50 ml. Embryos were allowed to sink and supernatant sea water was drained by gravity with a sump tube. A blue pipette tip was used as a thin inlet connected with the plastic tube (4 mm diameter) completely filled with sea water that was maintained by a metal clamp. This method worked very well in concentrating embryos into the volume of approximately 10 ml of sea water and polybrene was added (2 μg/ml). Flasks were placed horizontally to allow the embryos to be distributed over the larger area. Flasks were floated on the surface of a water bath maintained at 29 30°C. After one hour of incubation, flasks were placed vertically in the water bath and fresh sea water was added to 200 ml. The extra embryos were also incubated in the disposable plastic cup (200 ml) as an untreated control. Incubation was continued with aeration overnight.

The following day, about 10 13% of embryos hatched to nauplii in each of 4 vertical flasks and there was no significant difference with respect to the density during incubation with the vector (Table 2). None of the untreated group survived. Because the bottom of the flasks were in a water bath at 33°C, this may have adversely affected survival of the embryos.

Table 2

These results show that a one hour incubation with 40 embryos in 10 ml sea water in a 75 cm² flask is well tolerated by embryos. Embryos were collected, treated with ATA as described above, and 50-60 embryos in 200 ml sea water were aliquoted into each of three flasks. After the embryos had settled to the bottom, supernatant sea water was drained, leaving the embryos in 10 ml. At 45 mm post-spawning, three different viral vectors were added and incubated with polybrene (final cone. 2 μg/ml). The vectors were: 1 ) LNhsp70LucL, 2 x concentrated (1 x10⁸ cfu/ml x 20 μl); 2) 1 x concentrated bald virus x 100 μl; and 3) 1 x concentrated bald virus x 10 μl. After one hour incubation at 27°C, embryos were suspended in sea water as previously described. Approximately 50% of embryos hatched to nauplii in each group (Table 3).

Table 3

Pooled nauplii (n = 12) from the LNhsp70lucL group and from the bald virus (100 μl) group were analyzed by LTR PCR on day 3. Only the nauplii incubated with infectious vector were positive by PCR for the retroviral LTR in the vector. This confirms the successful infection of shrimp embryos with the pantropic retroviral vectors (Table 4).

Table 4

The remaining nauplii were fed and raised to zoea for individual PCR analysis. Four zoea incubated with LNhsp70lucL as embryos, and 6 zoea incubated with bald virus (100 μl) survived to day 7. They were individually placed into a 1.5 ml Eppeπdorf tube for DNA extraction and analyzed by PCR. One of the four zoea yielded a strong PCR signal, suggesting integration of the provirus early during development (possible transgemc embryo), and another zoea yielded a weak signal suggesting integration of the provirus later in development. None of the zoea from the bald virus group (negative control) was positive. All of the individual zoea were positive with actin PCR primers, confirming the presence of amplifiable DNA in all samples (Table 5). Table 5

Example 5 Somatic cell infection

Somatic infection with vectors carrying oncogeπes may lead to cell immortalization and creation of a persistent shrimp cell line. Four shrimps (approximately 10 g) were used. Since cell division is essential for retroviral infection, regeneration buds were created as described above. The retroviral vector LNhsplucL (2 μl containing 2 x 10⁵ cfu) was mixed with green food coloring (Durkee) and polybrene (2 μg/ml), and injected into the right-sided regeneration buds four days following the injury. 1 x concentrated bald viral vector (negative control) was injected into the left-sided regeneration buds. Five days after the vector injection, the regeneration bud was reamputated at the second distal joint and each tissue was individually analyzed by luciferase assay and LTR PCR (Jordan et al., supra.).

During injection, 20 60% of the vector with dye immediately drained to the body and did not remain at the site of injection. Nevertheless, 2/4 legs injected with LNhsp70lucL were positive by LTR PCR. All of the eight DNA samples extracted from each tissue were positive with actin PCR primers, verifying the quality of extracted DNA. The sensitivity of the LTR PCR on the same day was 30 copies of the provirai genome, suggesting that 2/4 regeneration buds had greater than 30 copies of provirai DNA. No luciferase activity was detected in any of the samples.

Greater than 30 proviruses integrated at the regeneration bud, suggesting successful induction of cell replication and retroviral infection. The absence of luciferase activity may be due to the low number of infected cells in each sample or to the poor function of the hsp70 promoter in shrimp.

In another experiment, the retroviral vector LNhsplucL (10 μl containing 1 x 10⁵ cfu) was mixed with green food coloring and polybrene (2 μg/ml) and injected into the heart of two living juvenile shrimp (2.5 g). Shrimp were harvested 3 days after infection. Tissues from different organs were analyzed by luciferase reporter gene expression and LTR PCR assay. DNA extracted from hemolymph and muscle was positive by nested PCR assay for LTR sequences, but heart, hepatopancreas and exoskeleton were negative. No luciferase expression was detected from any site. These results provide further evidence that retroviral vectors can infect and integrate into the genome of shrimp cells. These experiments provide the first documentation of in vivo somatic cell transformation in any shrimp species.

Example 6

Infection of thoracic organs

Intrathoracic injection of mature shrimp was performed to test infection of thoracic organs (ovary, Oka organ, heart) and muscles. Adult shrimp (50 60 g) were injected with LLRNL vector (10⁵-10⁶ cfu/shnmp) and assayed for luciferase expression at 3 11 days post-injection. The results are shown in Table 6. Expression of luciferase was observed in one of the shrimp for at least 11 days.

Table 6

Example 7

BrdU incorporation study in Oka organ primary culture

In order to obtain the maximum infection efficiency with retroviral vectors, it is important to expose primary cultured cells at the time of maximal cell proliferation. The cells in the S stage of mitosis (period of DNA synthesis before cell division) incorporate brommated deoxyundine (BrdU), which is easily detected by immunofluorescent staining with specific antibodies. At various times after starting Oka organ primary cell cultures (pronase dissociated),

BrdU was added to assess mitotic activity.

Oka organs were collected as previously described. Briefly, the Oka organ of adult shrimp (approximately 60g) was stimulated by injuring one of the peπopods. Shrimp were sterilized with serial immersions (2% sodium hydrochlonde, 1 % povidone iodine, and 95% ethanol). The Oka organ was dissected by a lateral approach. Cells were dissociated in 2 x L 15 with 20% FCS and 3% glucose using a sterile sieve (4 cm x 4 cm with 190 μm pore size), and seeded on Primaπa tissue culture plates. BrdU was added at specific times to the primary culture.

Staining for BrdU incorporation demonstrated that the mitosis in cultured Oka organ cells is most active within the first 6 hours of initiating the culture. Mitotic rates dropped at later timepoints. These results suggest that cells should be exposed to retroviral vector within the first 6 hours to maximize gene transfer.

Example 8

Gene expression in Oka organ primary culture The following examples described expression of foreign genes in primary cultured cells described from the

Oka organ and ovary of the penaid shrimp, P. stylirostris. However, it will be appreciated that foreign genes may also be expressed in cell lines from other species of shrimp, as well as in cell lines from other decapods, using the methods described herein.

Dissociated cells from the dissected Oka organ were seeded into 6-well tissue culture plates and exposed to the following retroviral vectors: 1) mock infection (no vector applied); 2) LLRNL (LTR luciferase-Rous sarcoma virus LTR-neomycm phosphotransferase-LTR); 3) LNGIAI LucL (LTR-neo-oyster actin promoter luciferase-LTR); and 4)

LNhsp70lucL (LTR Drosophi/a heat shock protein 70 promoter-luciferase-LTR. Cells were harvested at 72 hours post infection and luciferase activity was analyzed. The primary cultured cells exposed to LLRNL demonstrated high levels of luciferase gene expression (Fig. 2). Mock-infected, LNGIAI LucL and LNhsp70lucL infected cultures were negative (Fig. 2). This result shows that the MoMLV LTR functions as a good promoter in shrimp. To confirm the results from the previous experiment, LLRNL infection was repeated on the Oka organ primary cultures in triplicate. In addition, cells were exposed to LLRNL "bald" virus that lacks an envelope protein and is therefore non-infectious. The experiment was performed on Oka organs isolated from two individual shrimp. Luciferase expression was confirmed in the six primary cultures exposed to infectious, but not "bald", LLRNL (Fig. 3). The presence of the provirus (integrated vector) in these samples was confirmed by PCR.

Example 9 Primary cell culture from Oka organ and ovary The surface of a 60-70 g shrimp was decontaminated prior to dissection by serial immersions of shrimp in the following ice-cold solutions: a) 10% bleach x 5 mm., b) 1 % povidone iodine x 5 mm., and c) 70 % ethanol x 5 mm. A cell suspension was created by sieving the Oka organ or ovary through a stainless steel mesh (190 m pore size) into growth medium (2 x L 15 culture medium (Gibco BRL), 20% fetal calf serum, 100 g/ml streptomycin, 100 U/ml penicillin G, and 2.5 g/mi amphotencin B; osmolality: 670 mOsm). For the Oka organ only, the cell suspension was passed through a nylon mesh cell strainer (40 m pore size) before plating to remove debris. Cells were then seeded into 24-well plates (Costar) and grown at 28 C. From one 60-70 g shrimp, the Oka organ and ovary yielded sufficient cells to seed approximately 1-7 wells and 9 20 wells, respectively. For this reason, some experiments were performed with pooled Oka organ cells from several shrimp. This also served to reduce potential ammal-to-animai variation. Growth medium was replaced after 24 h and weekly thereafter.

Routine light microscopy was performed on histological sections of preserved ovary tissue and on primary cultured cells grown on a chambered slide (Lab-Tek, Nunc). Ovary tissue was excised as described above and a section of one lobe placed in Davidson s AFA fixative for 24 h (Lightener, A handbook of shrimp pathology and diagnostic procedures for disease of cultured penaeid shrimp, World Aquaculture Society, Baton Rouge, LA, p. 304, 1996). The tissue was then paraffin embedded and 4-5 m sections stained with hematoxyliπ and eosm according to standard methods (Lightner, supra.). Example 10

Infection with pantropic retroviral vectors

The pantropic vectors LNhsp70lucL, LN-IE-1-lucL, LLRNL, LNRLL, and LZRNL were produced as previously described (Figure 1A) (Jordan et al. supra.; Franco et al., Insect Biochem. Mol. Cell Biol. 28:28819 825, 1998; Xu et al., Virology 171:331 -341,1989). Pseudotyped vector particles were harvested from 293gag-pol (293gp) producer cell lines and concentrated by ultracentnfugation as previously described (Burns et al. supra.; Yee et al. supra.). Stocks of vector particles produced without an envelope glycoprotein ("bald" vector) are non-infectious and were prepared for use as negative controls (Teysset et al., J. Virol. 72:853-856, 1998) (Figure 1 B). Vector stocks were frozen at -70 C until use.

In different experiments, cells were infected with varying amounts of concentrated vector stock, from 0.1 -10 x 10^s cfu/well of a 24-well plate. Cultures were incubated with 250 I growth medium, 0 8 g/ml polybrene (poiycatioπ to overcome electrostatic repulsion between virus particle and cell surface), and vector overnight at 28 C. Negative control cultures were incubated with an equal volume of non-infectious, concentrated "bald" vector stock. Growth medium was replaced after overnight incubation.

Example 11

Assessment of mitotic activity

Mitotic activity was assessed in Oka organ cultures by incorporation of 5-bromo 2' deoxyuπdιne (BrdU).

Cultures were incubated with BrdU (10 M, Boehnnger Mannheim) at different intervals beginning 6 h post-initiation of cultures. Cells were fixed and stained with a BrdU antibody and FITC conjugated secondary antibody according to manufacturer's instructions at 30 h after addition of BrdU. Incorporation of BrdU was visualized by fluorescence microscopy and the number of FITC-labeled ceils estimated.

Staining Oka organ cultures with BrdU at intervals after initiation of the cultures helped to define the period of maximal cell division. Because vectors based on the munne leukemia viruses can only integrate into the genome of dividing cells when the nuclear membrane is disrupted, determination of the kinetics of cell division after initiation of the culture was used to establish the optimal timing for pantropic vector infection. Maximal BrdU incorporation was documented during the period from 6 24 h post-initiation of the culture, during which period between 10-15% of the cells were dividing.

Example 12

Provirai amplification and detection

To detect provirai DNA in cultured Oka organ cells, DNA was extracted on Culture Day 14 as previously described (Jordan et al., supra.). The pellet from extraction of one well of cells was resuspended in 100 I of TE buffer (1 mM Tns pH 7.5, 0.1 mM EDTA) and 1 I used for PCR amplification in a 25 I reaction with either the MoMLV LTR- specific primers or actin primers as previously described (Jordan et al. 1998). After 30 cycles, 10 I of the amplification product was loaded onto a 2% agarose gel and visualized by staining with ethidium bromide. Amplification of "bald" vector exposed cells was included as a negative control.

Example 13

Optimization of polybrene concentration in Oka organ primary culture The optimal concentration of polybrene was determined by infection in triplicate of cells derived from a pool of Oka organs. The polγcation, polybrene, is traditionally used to increase the infection efficiency of retroviral vectors by overcoming the net negative charge on the surface of the vector particle and the surface of the target cell. Addition of 0-8 g/ml of polybrene to the cultured cells at the time of infection with LLRNL showed no consistent effect of the polycation on infection efficiency. To determine if the lack of polybrene affect was cell-type specific, these experiments were repeated using cells derived from mature ovaries. Again, no benefit or toxicity of polybrene added to the culture supernatant could be demonstrated. Polybrene was therefore not used for subsequent experiments.

Example 14

Measurement of reporter gene expression

Cells for luciferase assay were harvested from wells at 72 h post-infection and washed once in 2X Hank's balanced salt solution. Washed cells were lysed in 70-120 I of cell lysis buffer (Analytical Luminescence Laboratory,

Sparks, MD). For assay of luciferase activity, 20-100 I of cell lysate were processed according to the manufacturer's instructions and light units measured in a Turner Design 20/20 lummometer. For protein assays, 4 I of cell lysate was analyzed with the Biorad Protein reagent as directed. For experiments in which heat shock was used to induce the

Drosophila hsp70 promoter, cells were incubated at 37 C for 1 h followed by 1 h at 28 C prior to lysing cells for the luciferase assay.

For detection of -galactosidase activity in cells infected with the LZRNL vector, monolayers grown on chambered slides were fixed according to the manufacturer's instructions (In situ -galactosidase Staining Kit,

Stratagene) and stained overnight with the X-gal staining solution.

In the first experiment, separate Oka organ cultures were established from two shrimp. Cells were either infected in triplicate with the vector, LLRNL, mock-infected, or exposed to "bald" vector. At 3-14 d post-infection, cells were lysed for reporter gene assay and PCR amplification of provirai LTR and actin sequences. Expression of firefly luciferase was easily and reproducibly detected in the LLRNL-mfected, but not "bald" vector exposed or mock infected cultures (Figure 5). Amplification with LTR-specific primers of DNA extracted from the nuclear pellets demonstrated the anticipated 244 bp amp coπ only from LLRNL-mfected cells. Amplification with the actin primers demonstrated the presence of amplifiable (inhibitor-free) DNA in every sample.

To determine which promoter would mediate the highest levels of luciferase expression, we compared four different vectors (Figure 1A) or "bald" LLRNL as a negative control (Figure 4). In three separate experiments, Oka organ primary cultures were prepared in 24 well plates from dissociated Oka organs of 1-3 shrimps, depending on the size of the organ. Each vector (10" or 10⁵ cfu/well) was applied to each well and luciferase activity was measured at 72 hours post infection All four vectors had an identical pseudotyped envelope, VSV-G, and differed only in the promoter that was used to express the luciferase reporter gene. LNIEIucL (luciferase gene expressed from Immediately-early promoter of insect baculovirus Autographa californica Multicapsid Nuclear Polyhedrosis Virus (AcMNPV)) and

LNhspLucL (luciferase gene expressed from Drosophila heat-shock 70 promoter) demonstrated no luciferase activity, suggesting that these promoters are not functional in shrimp cells. Luciferase activity from the wells infected with LNhsp70lucL could not be induced by heat-shock (37 °C for 1 hour, followed by 28 °C rest for 1 hour). LLRNL (luciferase gene expressed from MoMLV-LTR) expressed the highest luciferase activity and the negative control vector with same geπomic organization but with no infectious envelope (bald virus LLRNL) demonstrated no luciferase activity. LNRLL (luciferase gene expressed from Rous sarcoma virus LTR) demonstrated weaker luciferase activity than LLRNL. Three experiments were done independently and demonstrated consistent results. Therefore, the MoMLV-LTR is a strong promoter in shrimp cells.

A time course experiment with the vector LLRNL was performed to determine whether the duration and timing of contact with the vector would affect infection efficiency (Figure 5). Light units/ mg total cellular protein was used to estimate infection efficiency. Oka organ cells from three shrimp were incubated with retroviral vector LLRNL (10^s cfu/well) and polybrene (2 g/ml) for one or three hours at different intervals post infection of the culture. No toxic effect of the polybrene on the cells was observed by inspection of the monolayers. Therefore, all subsequent infections were performed within 2 h of seeding the wells and vector was incubated with the cells overnight. Three independent experiments were performed with LLRNL added to the cultures at three different time points after preparation of the Oka organ cultures. In Experiments 1 and 2, the cultured cells were derived from the Oka organ of a single shrimp/experiment, Experiment 3 was performed on pooled Oka organ cells from 2 shrimps.

To determine the relationship between vector dose and luciferase activity, Oka organ cells pooled from three shrimp were exposed in triplicate to three different concentrations of LLRNL vector and the luciferase activity in cell lysates determined at 72 h post-infection (Figure 6). An increase in viral titer from 10³ to 10" cfu/well resulted in a 44- fold increase in luciferase activity, while an increase from 10" to 10⁶ cfu/well resulted in only a 2.8 fold increase. Based on these studies, subsequent infections used 10 -10⁵ cfu/well.

To test whether other reporter genes could also be detected in transduced shrimp cells. Oka organ and ovary cells were infected with the vector LZRNL (Figure 1A). Cells were grown on chambered glass slides, infected with 10" cfu/chamber of LZRNL, and stained for -galactosidase expression at 72 h post-infection. For the cultured ovary cells, efforts were made to use cells from the mid-section of the gland to avoid introducing chromophore cells (dark blue- black in color) into the cultures. Clumps of blue-stained cells were apparent in LZRNL-mfected cultures from both organs but not in mock-infected control cultures.. Thus, β-galactosidase is present in retrovirally transduced cells. Example 15

Embryo primary cell culture

Embryo primary cell culture is another source for a transformed cell line. These cells are actively dividing and the choπon surrounding the cells serves as a protective barrier against the toxic effects of chemical sterilization.

Embryos (about 3 4 hours old) were collected with a nylon mesh and sterilized with either formalin, sodium hypochloπte, povidone iodine, ethanol, or different combinations of these agents. Embryos were dissociated mechanically with a pestle and tested for both microbial contamination and cell viability, which was assessed by acndine orange vital fluorescence and ethidium bromide exclusion fluorescence. The dissociated cells were observed in various concentrations of sea water or culture media to determine the optimal environment.

Sodium hypochlonte, ethanol and formalin at the effective concentration against bacterial contamination demonstrated toxicity to the cells. Iodine 1 :10 dilution (1 %) in sea water for 1 mm eliminated bacterial contamination without any recognizable cell toxicity compared to non-treated cells. Cell viability was confirmed by bright vital staining of acndine orange and the culture was observed for four days. The fluorescent staining with acndine orange and ethidium bromide identified live and dead cells very clearly. Occasional protozoa! contamination occurred. The organism is susceptible to formalin 1:100 dilution which, itself, is not concentrated enough to eliminate bacteria, but shows no toxicity to the embryo cells. Thus, 1 % iodine and 1:100 formalin may be the best sterilization technique with minimal toxicity. The cells were easily dissociated mechanically by pestle and did not aggregate during incubation in the media. Dissociated cells survived equally well in various concentrations of seawater ranging from 650-1030 osmolanty (Osm). The dissociated embryonic cells tested in three different media demonstrated the best survival in the mixture of sterile seawater and 2 x L-15 (1 :1 ) (850 Osm), compared to 1 x L15 in 0.9 seawater (adjusted to be isotomc to seawater, 1030 Osm), or 2 x L15 (650 Osm). Thus, dissociated embryonic cells are another suitable target for retroviral infection.

It should be noted that the present invention is not limited to only those embodiments described in the Detailed Description. Any embodiment which retains the spirit of the present invention should be considered to be within its scope. However, the invention is only limited by the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for introducing exogenous DNA into a decapod, comprising: infecting a decapod embryo with a retroviral pseudotype comprising said exogenous DNA, said retroviral pseudotype comprising: retroviral long terminal repeats (LTRs);

RNA corresponding to a promoter within said LTRs;

RNA corresponding to said exogenous DNA, wherein said exogenous DNA is operably linked to said promoter; and

VSV-G protein encapsidating said RNA.

2. The method of Claim 1, wherein said retroviral pseudotype is prepared by: inserting said exogenous DNA into a first plasmid comprising said retroviral LTRs and said promoter such that said exogenous DNA is operably linked to said promoter; and forming said retroviral pseudotype by expressing said plasmid in a cell comprising a VSV-G gene and retroviral gag and pol genes.

3. The method of Claim 1 , wherein said LTRs are MoMLV LTRs.

4. The method of Claim 2, wherein said plasmid comprises a selectable marker.

5. The method of Claim 1, wherein said decapod is a shrimp.

6. The method of Claim 2, wherein in the forming step comprises: transfecting a packaging cell line with said first plasmid, wherein said packaging cell line assembles retroviral particles comprising an RNA copy of said exogenous DNA; infecting a producer cell line with said retroviral particles, said producer cell line comprising said retroviral gag and pol genes; and transfecting said vector particle-containing producer cell line with a second plasmid containing a promoter operably linked to said VSV-G protein, whereby pseudotyped retroviral vectors containing VSV-G protein are secreted by said producer cell line.

7. The method of Claim 6, wherein said packaging cell line is PA317.

8. The method of Claim 6, wherein said producer ceil line is 293 cells.

9. The method of Claim 6, wherein said second plasmid is pHCMV-G.

10. The method of Claim 6, wherein said promoter of said second plasmid is cytomegaiovirus promoter.

1 1. The method of Claim 1, additionally comprising dechorionating said embryo of said decapod prior to infecting.

12. A method for introducing exogenous DNA into a decapod cell line, comprising: infecting a decapod cell with a retroviral pseudotype comprising said exogenous DNA, said retroviral pseudotype comprising: retroviral long terminal repeats (LTRs); RNA corresponding to a promoter within said LTRs;

VSV-G protein eπcapsidating said RNA.

13. The method of Claim 12, wherein said retroviral pseudotype is prepared by: inserting said exogenous DNA into a first plasmid comprising said retroviral LTRs and said promoter such that said exogenous DNA is operably linked to said promoter; and forming said retroviral pseudotype by expressing said plasmid in a cell comprising a VSV G gene and retroviral gag and pol genes.

14. The method of Claim 12, wherein said decapod is a shrimp.

15. A decapod expressing exogenous DNA.

16. The decapod of Claim 15, wherein said decapod is a shrimp.

17. A decapod cell line expressing exogenous DNA.

18. The decapod cell line of Claim 17, wherein said decapod is a shrimp.