Sandra L. Ayres

Production of goats by somatic cell nuclear transfer

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

Adeno-associated virus (AAV)-mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation

We explored whether exposure of mammalian germ line stem cells to adeno‐associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell‐depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild‐type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV‐transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV‐mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.—Honaramooz, A., Megee, S., Zeng, W., Destrempes, M.M., Overton, S.A., Luo, J., Galantino‐Homer, H., Modelski, M., Chen, F., Blash, S., Melican, D. T., Gavin, W. G., Ayres, S., Yang, F., Wang, P. J., Echelard, Y., Dobrinski, I. Adeno‐associated virus (AAV) ‐mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J. 22, 374–382 (2008)

Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation†

Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor‐derived transgenic sperm. The objectives of this study were to explore a non‐viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8–9 weeks post‐irradiation. Donor cells were collected from testes of 9‐week‐old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta‐casein promoter (GBC) and a chicken beta‐globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non‐transfected cells to a total of 1.5 × 108 cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound‐guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof‐of‐concept that non‐viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255–261, 2012.

Induction of HIV-1 MPR649–684-specific IgA and IgG antibodies in caprine colostrum using a peptide-based vaccine☆

Induction of antigen-specific antibodies against HIV-1 in colostrum and milk may help prevent breast milk transmission of the virus. A peptide vaccine against the HIV-1 gp41 membrane proximal region (MPR(649-684)) was evaluated as proof-of-principle in a caprine model. Pregnant Alpine/Saanen goats were immunized with MPR(649-684) peptide conjugated to KLH using alum adjuvant. Immunizations were intramuscular, intranasal, and in the supramammary lymph node region. Samples collected after parturition demonstrated the presence of MPR(649-684)-specific antibodies in colostrum and serum. These results support the concept that a peptide vaccine can effectively induce MPR(649-684)-specific sIgA and IgG in the colostrum of a lactating species.

Comparison of indocyanine green and sodium fluorescein for anterior segment angiography of ophthalmically normal eyes of goats, sheep, and alpacas performed with a digital single-lens reflex camera adaptor

OBJECTIVE To compare results of anterior segment angiography of ophthalmically normal eyes of goats, sheep, and alpacas performed by use of indocyanine green (ICG) and sodium fluorescein (SF). ANIMALS 10 female goats (mean ± SD age, 6.8 ± 1.7 years), 10 female sheep (3.0 ± 2.2 years), and 10 alpacas (7 females and 3 males; 6.8 ± 3.8 years). PROCEDURES A catheter was aseptically placed into a jugular vein. Each animal was anesthetized and properly positioned, and 0.25% ICG was administered. Images were obtained by use of an adaptor system consisting of a modified digital single-lens reflex camera, camera adaptor, and camera lens. Images were obtained at a rate of 3 images/s for the 60 seconds immediately after ICG administration and then at 2, 3, 4, and 5 minutes after administration. Ten minutes later, 10% SF was administered IV and images were obtained in a similar manner. RESULTS Angiography with ICG provided visual examination of the arterial, capillary, and venous phases in all species. Visual examination of the iris vasculature by use of SF was performed in goats and sheep but was not possible in the alpacas because of iridal pigmentation. Extravasation of SF was a common finding in sheep and alpacas but not in goats. No adverse events were detected. CONCLUSIONS AND CLINICAL RELEVANCE Quality angiographic images of the anterior segment were obtainable after IV administration of ICG to goats, sheep, and alpacas. This may provide a useful imaging modality for ocular research in these animal species.

Surgical management of the goat.

potential zoonotic diseases that may be transmitted by the goat are mainly of concern during particular events, such as abortion or parturition. More importantly, goats may carry a number of potential zoonotic diseases3 that may be transmitted to humans at a higher frequency than is Q fever4, including infections with Chlamydia5, Campylobacter6, Listeria7 and Leptospira8. Isolating Q fever as a primary zoonotic concern when handling goats reflects a historic emphasis resulting from isolated incidents that were highlighted in the literature many years ago and continue to get particular attention and reference9,10. We feel that if goats are acquired from good-quality dealers and vendors, undergo specific pathogen–free testing (including testing for Q fever) both before procurement and while they are at the research facility11 and are quarantined upon arrival, the risk of zoonotic Q fever transmission can be reduced to a very low and acceptable level, compared with the risk of zoonotic transmission of other agents that goats may harbor. In addition, goats are not the sole vector for Q fever. The existing literature suggests that people other than those working with animals or those who live or work in agricultural settings can be exposed to Q fever12,13. Research has shown that people in urban settings have been exposed to Q fever in their normal environments13. In the US, there have been no widespread outbreaks of Q fever resulting from exposures in the agricultural environments or through direct contact with ruminants, other animals or other known vectors for Q fever11,14–16. There is a low incidence of Q fever in the US; according to information on the Centers for Disease Control website (http://www.bt.cdc.gov/coca/), fewer than 200 cases of Q fever are reported annually in the US. When working with goats personnel should, at a minimum, use basic precautions. This may include PPE such as gloves, laboratory coats or surgical scrubs, in accordance with the accepted practice at a particular research facility. Special attention should also be given to hand-washing and other standard practices such as prohibiting food or drinks in laboratory or animal settings. Personnel should be aware that many zoonotic diseases may be carried in the goat as part of its normal flora and fauna, as is discussed in the Guide for the Care and Use of Laboratory Animals17 and the Guide for the Care and Use of Agricultural Animals in Research and Teaching18. Personnel changing bedding or attending births should wear gloves and coveralls or surgical scrubs. There have been no cases of zoonotic Q fever transmission to any of our personnel. Additionally, a number of staff members have chosen to continue working with goats on a daily basis during their pregnancies and have delivered healthy children without incident. Third, we feel that shaving a goat’s abdomen and placing a gastrostomy tube in a goat before surgery to address stomach build-up of gas or fluid is unnecessary. This is not standard practice in our group and may be viewed as an unneeded and Surgical management of the goat