Carol Ziomek

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.

Commercialization of proteins produced in the mammary gland

In the mid 1980s, few pioneering companies undertook the risk of developing methodologies for the production of complex human therapeutic proteins in the milk of transgenic animals. As we approach the end of the 1990s, the prospect of achieving this aim is becoming a reality as the first of these human therapeutic products, antithrombin III and alpha-1-antitrypsin are making their way through human clinical trials. It is projected that licensure by the Regulatory agencies and market launch for these transgenically produced therapeutics will occur around the year 2000. Although much has already been achieved, additional transgenic challenges await the basic embryo researcher and practitioner. The biopharming community recognizes the need for additional innovative methodologies (such as cloning, sperm sexing and retroviral mediated gene transfer etc.) to overcome the natural biological barriers and increase the efficiency of transgenic dairy animal production and rapid herd expansion.

Transvaginal ultrasound-guided oocyte retrieval following FSH stimulation of domestic goats☆

The objectives of this study were to evaluate different ovarian stimulation protocols on donor goats and to develop a safe, repeatable method for harvesting oocytes from FSH-treated does (Experiment I). Based on the preliminary findings of the first experiment, 32 crossbred does were used in a second experiment (Experiment II), 16 that had not been previously aspirated and 16 that had undergone one previous aspiration, were used to fine tune the procedure. Females were randomly subjected to 1 of the 2 ovarian stimulation protocols: Treatment (A) does were implanted with a norgestomet ear implant. Starting 10 d post-implantation, does were administered FSH daily for 4 d. Does in Treatment (B) were treated similarly to those in (A) but were implanted for only 3 d before starting the FSH injections and implants were not removed prior to aspiration. Using a 2 x 2 factorial arrangement, fresh does (n=16), not previously aspirated, were then further randomly assigned to either a laparoscopic aspiration procedure (LAP) or a transvaginal ultrasound-guided aspiration procedure (TUGA). The LAP procedure was performed using a fiber optics. For the TUGA, the doe was placed in dorsal recumbency, and a 5 MHz human transvaginal transducer, attached to the ultrasound unit, was positioned vaginally for oocyte aspiration. In summary, there was no significant difference among treatment groups for parameters evaluated, with the exception of methods for oocyte collection. The number of follicles detected and oocytes harvested using TUGA (9.5 and 4.3, respectively) was less than for females obtained by LAP (17.4 and 14.4, respectfully). The percentage of oocytes recovered from does subjected to the TUGA (68%), however, was similar to those subjected to the LAP (69%). Unlike donor does subjected to a repeated LAP, there was no evidence of adhesions in donor does from the repeated TUGA group. The TUGA approach to oocyte collection should not be overlooked in an effort to decrease the chances of adhesions in valuable donor goats.

Hormonal induced lactation in transgenic goats

Abstract The aim of this study was to hormonally induce lactation in prepubertal, nulliparous, and male goats both transgenic and non‐transgenic. Analysis of milk quality, recombinant protein expression levels, total amount of recombinant protein produced, and the affect on long‐term reproductive capability was assessed. Fifty‐one goats (Saanen, Alpine, and Toggenburg), male and non‐pregnant females, 2‐31 months of age, either non‐transgenic or transgenic were evaluated with a total of 10 transgenes (constructs) represented. Animals were given estradiol (0.25 mg/kg, IM) and progesterone (0.75 mg/kg, IM) on days 1, 3, 5, 7, 9, 11 and 13, while prednisilone (0.4 mg/kg, IM) was administered on days 14‐16 with mammary massage occurring daily from day 5 onward. Forty of 51 animals, (36 of 38 females and 4 of 13 males) produced milk with total volumes in the 30‐day experiment, ranging from 20 μl to 530 mls per day, or approximately 500 ul to 6.8 liters total. Milk composition was analyzed for various parameters (total protein, fat content, total solids and somatic cell count) with no significant differences found between induced and natural milk. Expression levels of recombinant proteins from transgenic animals that were analyzed during the induced lactation, and subsequently during normal lactations, were found to have no significant differences. Total amount of recombinant protein produced was evaluated at different expression levels with no statistical significance seen. While over 90% of the females placed in the regimen became pregnant, there was a correlation between increased age at time of induction and an increase in number of breedings, or reproductive cycles needed to establish a pregnancy after induction. For males, 100% placed in the regimen settled females after hormonal induction of lactation. Semen quality was evaluated prior to, during, and after hormonal treatments. Semen volume and sperm number did not differ; however, for a small percentage of males, there was a decrease in sperm and post thaw motility after hormonal treatments. These levels returned to normal within 4‐5 weeks. Subsequent natural lactations showed total milk volumes within breed standards. These findings indicate that hormonal induction of lactation in the caprine species is a viable alternative to pregnancy for initiating lactation and milk production, does not adversely impact reproductive performance long‐term, and can benefit the early assessment of recombinant proteins produced in a transgenic founder program.

Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos.

Glutamine is utilized as an energy substrate in preimplantation mouse embryos. Glutaminase is the enzyme responsible for the conversion of glutamine to glutamic acid, which then enters the trichloro acetic acid (TCA) cycle as α‐ketoglutarate. Glutaminase enzyme activity was assessed in preimplantation embryos that developed in vivo, and glutaminase RNA expression was examined in embryos that developed in vivo or were cultured in CZB medium to various preimplantation stages between 1‐cell and blastocyst. Glutaminase activity in 1–8‐cell‐stage mouse embryos that developed in vivo ranged from 0.009–0.01 U/mg protein (2.39–2.95 × 10−7 U per embryo), and increased 3–4‐fold to 0.034 U/mg protein (8.13 × 10−7 U per embryo) at the blastocyst stage. Relative stage‐specific expression of glutaminase RNA was assessed by reverse transcription polymerase chain reaction (RT‐PCR) in embryos that developed both in vivo and in CZB culture. In vivo, glutaminase RNA was expressed at the 1‐cell stage, declined to 23% of 1‐cell levels at the early 2‐cell stage, and reaccumulated from late 2‐cell through blastocyst stage, where it reached a high of 204% of 1‐cell levels. CZB‐cultured embryos exhibited a similar pattern of developmental RNA expression, declining to 30% of 1‐cell levels at the early 2‐cell stage, and increasing RNA expression at the blastocyst stage to 191% of the 1‐cell level. Mol. Reprod. Dev. 47:248–254, 1997.