Regulatory Dysfunction inhibits the Development and Application of Transgenic Livestock for Use in Agriculture

It has been 30 years since the first transgenic mouse was generated and 26 years since the first example of transferring the technology to livestock was published. While there was tremendous optimism in those initial years, with most convinced that genetically modified animals would play a significant role in agricultural production, that has not come to be. So at first sight one could conclude that this technology has, to a large extent, failed. On the contrary, it is believed that it has succeeded beyond our original expectations, and we are now at what is perhaps the most exciting time in the development and implementation of these technologies. The original goals, however, have drastically changed and it is now biomedical applications that are playing a central role in pushing both technical and scientific developments. The combination of advances in somatic cell nuclear transfer, the development of induced pluripotent stem cells and the completion of the sequencing of most livestock genomes ensures a bright and exciting future for this field, not only in livestock but also in companion animal species.

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Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5473-5479.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5473-5479

Author(s):

C M Shanahan ◽

N W Rigby ◽

J D Murray ◽

J T Marshall ◽

C A Townrow ◽

...

Keyword(s):

Growth Hormone ◽

Transgenic Mice ◽

Mrna Expression ◽

Transgene Expression ◽

Fusion Gene ◽

Developmental Expression ◽

Regulation Of Expression ◽

Tissue Specific ◽

Transgenic Livestock ◽

Main Regulator

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.

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Techniques for the Production of Transgenic Livestock

10.1201/9781315137483-12 ◽

2021 ◽

pp. 167-181

Author(s):

W.H. Eyestone

Keyword(s):

Transgenic Livestock

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Transgenic Livestock Technologies, Emerging

10.1007/springerreference_226442 ◽

2012 ◽

Keyword(s):

Transgenic Livestock

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5 PRODUCTION OF TRANSGENIC LIVESTOCK USING A LENTIVIRUS EXPRESSING MULTIPLE SHORT INTERFERING RNAs TARGETING FOOT AND MOUTH DISEASE VIRUS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab5 ◽

2011 ◽

Vol 23 (1) ◽

pp. 109

Author(s):

M. Peoples ◽

M. Westhusin ◽

K. Tessanne ◽

C. Long

Keyword(s):

Disease Virus ◽

Lentiviral Vector ◽

Tissue Sample ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Antibiotic Selection ◽

Viral Particles ◽

Transgenic Livestock ◽

Mouth Disease Virus

One goal of transgenic livestock production is developing animals with enhanced production characteristics. Transgenic animals with resistance to viral disease could greatly reduce economic losses. The use of short interfering RNA (siRNA) or short hairpin RNA (shRNA) targeting viral genomes have shown great promise in vitro for both human and animal applications. However, because of the rapid mutation rate, viruses are able to escape single siRNA inhibition. One method to reduce the chances of a functional escape virus is to target its genome with multiple shRNAs simultaneously. The goal of this research project was to produce a recombinant lentiviral vector that expresses three unique shRNAs targeting different regions of the foot and mouth disease virus (FMDV) and use it to produce transgenic livestock. In these initial experiments we used the goat as our model system. Previously, we confirmed that three distinct siRNAs individually could reduce the ability of the FMDV virion to replicate in vitro. Based upon these results we produced a recombinant lentiviral vector that utilised three bovine Pol III promoters (7sk, H1, U6-2), each transcribing a different effective shRNA targeting FMDV. In addition, the vector also contained the fluorescent marker zsGreen and an antibiotic resistance gene. The lentiviral vector was co-transfected with pCMV and pMDG into 293T cells to produce replication incompetent retroviral particles. The supernatant was collected and ultra-centrifuged (50 000 × g for 1.5 h) to concentrate the viral particles resulting in a high-titer viral preparation (>109 mL–1 infective viral particles). To produce the transgenic caprine offspring, three embryo donors were superovulated and naturally bred. Nineteen zygotes were surgically collected from the oviduct 24 h after mating. Recombinant lentivirus was microinjected into the zygote perivitelline space. Immediately following the injections, four goat embryos were surgically transferred into the oviduct of each synchronized recipient. Pregnancy was determined by ultrasound at Day 30 in 2 of 5 recipients that received embryos. One pregnancy was carried to term resulting in triplets; 1 live birth, and 2 stillborn. The placenta and tissue sample of the live goat both contained a subpopulation of zsGreen positive cells when analysed with fluorescent microscopy. A fibroblast cell line was derived from the tissue sample and placed under antibiotic selection. Results indicate that only the fluorescent cells also expressed a resistance to antibiotic selection. RNA was collected from the fibroblast cells and mature shRNA production was confirmed using the QuantiMir kit (System Biosystems). Expression of all 3 mature shRNAs was verified in these cells. This data further supports that the entire transgene was integrated into the genome. This is the first report of transgenic livestock produced that expresses multiple shRNAs targeting a viral genome.

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436 RISK ANALYSIS OF α-LACTALBUMIN TRANSGENE TRANSFER TO NONTRANSGENIC CONTROL ANIMALS DURING REARING, BREEDING, PARTURITION AND LACTATION

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab436 ◽

2010 ◽

Vol 22 (1) ◽

pp. 375 ◽

Cited By ~ 1

Author(s):

M. B. Wheeler ◽

W. L. Hurley ◽

J. Mosley ◽

G. E. Bressner ◽

E. Monaco ◽

...

Keyword(s):

Mammary Gland ◽

Close Contact ◽

Initial Step ◽

Swine Production ◽

Physical Conditions ◽

Bodily Fluids ◽

Transgenic Livestock ◽

Lactating Mammary Gland ◽

Placental Membranes ◽

Transgenic Swine

Assessment of general risk posed from transgenic (T) animals is important to their future contributions to society. Identification of potentially harmful properties of transgenic livestock is the initial step in a risk assessment. We previously developed and characterized transgenic swine containing a mammary-specific transgene (bovine a-lactalbumin, bALAC) that results in increased milk production in sows. We are currently determining whether bALAC is expressed in tissues of T swine other than the lactating mammary gland and whether the transgene DNA (Tg) crosses into nontransgenic control (C) swine under various physiological and physical conditions. The specific aims addressed in the present study were to determine (1) whether the Tg can be transferred directly by physical association or contact; (2) whether the Tg can be transferred directly via mating; (3) whether the Tg can be transferred directly during gestation and parturition; and (4) whether the Tg can be transferred directly during lactation. The T animals utilized in these studies are in at least generation 10 and have stable incorporation of the Tg. Comparable age- and weight-matched animals, T and C, were housed together allowing general contact that is normal in swine production, for either 180, 220, or 250 days of age after weaning. Swine typically ingest saliva, regurgitated food, and stool or urinary products, as well as other bodily fluids and cells during normal housing. In the second study, vaginal, cervical, uterine, oviductal, and ovarian tissues from C females on 2, 7, or 90 days after mating to T males, and penis, bulbourethral gland, urethra, testis, and epididymis tissues from C males on 2 or 7 days after mating to Tg females were collected. The presence of Tg in tissues from all C animals was tested by using PCR. We have analyzed for the presence of the Tg in various tissues [including mammary gland, salivary gland, skin (sebaceous gland), muscle, lung, liver, kidney, brain, ovary, oviduct, uterus, cervix, vagina, penis, bulbourethral gland, urethra, testis, epididymis, blood, inner and outer placental membranes and intestine]. Results indicate no presence of the Tg in tissues of C animals (n = 28) after co-habitation for 180, 220, or 250 days (n = 305 samples analyzed) or at 2 (n = 7), 7 (n = 16), or 90 (n = 6) days post-mating (n = 72, 192, or 71 samples analyzed, respectively). At Day 112 of gestation, all the samples (n = 78 samples analyzed) from nontransgenic piglets (n = 13) whose dam was aTg female were negative except for the outer placental membrane (n = 13), which screened positive for the transgene. This is not surprising because the outer placental membrane is in close contact with the uterus of the Tg dam. Finally, control piglets (n = 4) that were cross-fostered (3 days after birth) and suckled Tg dams showed no evidence of the transgene in their tissues (n = 20 samples analyzed) at weaning. The present results suggest that there is no horizontal Tg transmission between T and C pigs caused by rearing, mating, gestation, or lactation. This project was supported by USDA BRAG Project #2005-03799.

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337 PRODUCTION OF TRANSGENIC SHEEP USING RECOMBINANT LENTIVIRUS MICROINJECTION OF IN VIVO PRODUCED EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab337 ◽

2011 ◽

Vol 23 (1) ◽

pp. 264 ◽

Cited By ~ 1

Author(s):

K. Tessanne ◽

C. Long ◽

T. Spencer ◽

C. Satterfield ◽

M. Westhusin

Keyword(s):

Genomic Dna ◽

Viral Vectors ◽

Effective Means ◽

Alternative Methods ◽

Pcr Analysis ◽

Perivitelline Space ◽

Viral Supernatant ◽

Transgenic Livestock ◽

Transgenic Sheep

The development of transgenic technology has enormous potential for livestock improvement. Several methods have been employed for generating transgenic livestock to date, the most popular being pronuclear injection or somatic cell nuclear transfer using genetically modified cells. However, the high cost and relatively low efficiencies seen with these methods have prompted development of alternative methods for producing transgenic livestock. Recently, the introduction of transgenes using viral vectors, in particular lentiviral vectors, has provided an avenue for increasing the efficiency of transgenic livestock production. Microinjection of concentrated lentivirus into the perivitelline space of oocytes and zygotes has been demonstrated as an effective means of creating transgenic livestock (cattle, sheep, and swine). In this study, we investigated the efficiency of producing transgenic sheep through microinjection of recombinant lentivirus into in vivo produced zygotes. Recombinant lentivirus was produced through co-transfection of HEK293T cells with various lentiviral transfer plasmids (each coding for a short hairpin RNA and a fluorescent marker protein) as well as a packaging plasmid and a plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), which was used to pseudotype viral particles. For lentivirus concentration, a total of 36 mL of viral supernatant was produced per viral construct. Viral supernatant was ultracentrifuged on a 15% sucrose cushion at 4°C for 1.5 h at a speed of 50 000 × g. The resulting viral pellet was resuspended in 30 μL of PBS and virus was frozen at –80°C until needed. Zygotes were surgically flushed from oviducts of superovulated donor ewes 24 h post-mating. Concentrated recombinant lentivirus (titer ≥ IU mL–1) was then microinjected into the perivitelline space. Injected embryos were surgically transferred into the oviduct of synchronized recipient ewes immediately after injection (3–4 embryos per ewe). Pregnancies were confirmed by ultrasound at 35 days of gestation. The pregnancy rate was 39% (45/114 ewes exhibiting at least one viable fetus). A subset of pregnant ewes was killed at 60 to 70 days gestation to harvest a total of 36 fetuses. Analysis of transgene incorporation was performed by PCR using genomic DNA isolated from skin and liver tissue samples. Two independent PCR reactions were performed per sample, and PCR analysis revealed 14 of the collected fetuses to be transgenic (39%). The remaining pregnancies were allowed to progress to term, and 32 lambs were born. Genomic DNA was isolated from blood samples collected on each lamb, and PCR analysis was performed as above. Of the lambs born, 13 of 32 (40%) were confirmed to be transgenic by PCR analysis. Southern blot analysis is currently underway to confirm PCR data. These results demonstrate that microinjection of recombinant lentivirus into in vivo produced sheep zygotes is an effective and efficient method for generating transgenic sheep.

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