94 Impact of number of embryos transferred on the number of offspring produced in a commercial transgenic founder production operation

The production of transgenic founder dairy goats (cross-bred Saanens, Alpines, Toggenburgs, and Nubians) involves the collection, microinjection, and transfer of numerous embryos into suitable recipient goats to ultimately produce a transgenic founder(s). The objective of this study was to determine the most efficient number of microinjection embryos to transfer to suitable recipients for transgenic founder generation. This is critically important in a commercial production program, as it impacts the goal for the number of embryos collected from donors, number of recipients utilised, and, hence, the overall number of surgical procedures being performed. The entire embryo collection, transfer, and founder-generation process is continuously being evaluated for ways to become more efficient in producing transgenic animals. During LFB USA’s commercial founder-production campaigns over the years (1997-2017), pronuclear microinjection was performed and 3, 4, or 5 embryos were transferred to female goat recipients. The recipients were synchronized using a progesterone implant on Day 0, a prostaglandin injection at Day 7, an injection of 300-500IU of pregnant mare serum gonadotropin on Day 13, progesterone implant removal on Day 14, and surgical transfer of pronuclear microinjected 1- or 2-cell embryos into the oviduct on Day 17. The individual totals and calculation for offspring per embryos transferred was compared for 3, 4, and 5 embryos transferred per recipient and was determined to be (1659/8637) 0.19, (912/4548) 0.20, and (112/675) 0.17, respectively. These embryo efficacy ratios were not significantly different (P>0.05) using the Wald Chi-squared test under logistic regression, and suggests that the number of offspring born is not impacted by number of embryos transferred. Seasonality was also evaluated in this production environment located in North America, with in-season being considered September to December and out-of-season being January to July. Nulliparous recipients during in-season (September to December) embryo transfer operations produced a significant difference, with totals and calculation for (offspring per embryo transferred) of (470/2346) 0.20, (260/1088) 0.24, and (23/190) 0.12 for 3, 4, and 5 embryo transfers, respectively (Table 1). This data indicates that when using nulliparous recipients during the in-season, transferring 4 embryos is optimal for offspring produced. Table 1.Comparison of the individual totals and the calculation of (offspring/embryo) by parity and season

Generation of a Transgenic Mouse with Inducible Constitutively Active Stat5a,

Abstract 3399 The Jak/Stat signalling pathway is essential for survival and proliferation of haematopoietic cells. The Stat5 transcription factors (Stat5a and Stat5b) play a crucial role in the development of various lineages of the hematopoietic system. Stat5 is activated by many cytokines and growth factors that regulate hematopoiesis. Persistent Stat5 activity is frequently found in hematopoietic cancers due to aberrant tyrosine kinase signalling. We have previously characterized a constitutively activated version of Stat5 (called oncogenic Stat5; cS5F), which mimics persistent tyrosine kinase signalling and promotes multi-lineage leukaemia development in a bone marrow transplantation model. The aim of the study is to generate a novel inducible mouse model for oncogenic Stat5a using the BAC recombineering technology. A cassette expressing cS5F (with a C-terminal FLAG tag) and a reporter gene (truncated human CD2; hCD2) was cloned, sequenced and biochemically verified for persistent tyrosine phosphorylation in absence of cytokines or growth factors. The cassette is flanked by loxP sites in opposite orientation and was used to replace the original Stat5a gene in a BAC carrying the endogenous Stat3/5 locus using BAC recombineering. Therefore, the expression of the transgene is regulated by the endogenous Stat5a promoter. The transgene is in an inverted (off) orientation and can be switched into the transcribing (on) orientation by Cre activity. Pronuclear injections were performed with the linearized and purified BAC. We obtained 5 transgenic founder lines which showed germ line transmission of the transgene. The copy number of the BAC was estimated to be 2 by Southern blot analysis. The transgenic founder lines were bred with the Rosa CreERT2 mice, and the Cre activity was induced by treating the mice with 1 mg of tamoxifen every day, for 5 consecutive days. The recombination of the transgene into the ‘on' orientation in liver, lungs, kidney, spleen and bone marrow was demonstrated by Southern blotting. The expression of the hCD2 marker was detected in various hematopoietic lineages by FACS and the expression of the transgenic protein in liver was confirmed by Western blotting with anti-Stat5a and anti-FLAG antibodies. One week after induction of the transgene, all the mice induced with tamoxifen (n=6) developed atrophic thymii with nearly a 4-fold reduction in total thymocyte numbers (p=0.0003). However, the percentage of CD8+ cells was increased more than 3 fold in the thymus (p=0.0002). Moreover, there was a 2-fold reduction in the number of early hematopoietic progenitors; defined as lin−, Sca1+ and c-kit+, in the bone marrow (n=5, p=0.0217). We have established an inducible mouse model for expression of constitutively active Stat5a under the endogenous promoter. We are currently monitoring these mice for development of cancer as they provide a competent tool to study the molecular mechanisms triggered by persistent Stat5a activity, including identification of other cooperating signalling pathways that contribute to generation of cancer. Moreover, it would allow one to test interference strategies, which could lead to potential therapeutics. Disclosures: No relevant conflicts of interest to declare.

432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.

61 GENERATING A HERD OF TRANSGENIC DAIRY GOATS BY SOMATIC CELL NUCLEAR TRANSFER

Transgenic dairy goats expressing recombinant therapeutic molecules in the milk may offer an alternative manufacturing method compared to traditional cell culture. Typically, a herd of transgenic progeny can be efficiently generated by naturally mating a transgenic buck with multiple does. However, the ability to rapidly generate a herd of transgenic progeny from a female transgenic founder by natural mating represents a challenge. While a herd of transgenic progeny can be generated from a female transgenic founder by superovulation and either in vitro fertilization or embryo flushing, the time period required is dependent on the number and gender of progeny needed. In this work we determined the ability of using skin cells from two transgenic founder female dairy goats as karyoplasts for nuclear transfer to produce a herd of transgenic female dairy goats. Two transgenic founder female dairy goats expressing a recombinant therapeutic human protein in the milk were generated by nuclear transfer using a transfected fetal cell line. Primary cultures of transgenic skin cells were established from these two founder lines using tissue samples obtained by biopsy. The cells were used as nuclear donors following three or four days of low-serum culture [0.5% fetal bovine serum (FBS)]. Donor cells were simultaneously fused and activated with in vivo produced oocytes from superovulated does. Nuclear transfer embryos were transferred to synchronized surrogate recipients at 24 to 48 h post-fusion and activation. Pregnancies were determined by ultrasonography starting at approximately Day 28 post-fusion and activation and then monitored weekly. The results of work conducted during two defined breeding periods are summarized in Table 1. While there were no significant differences in the number of Day 50 pregnancies (17 vs. 27), there were significantly more term pregnancies (14 vs. 12) and offspring produced (20 vs. 15) during the traditional (September through December) compared with the nontraditional (January through May) breeding seasons, respectively (P < 0.05). In this work, 35 female NT offspring were produced, requiring 13 months from the start until the last offspring was born. These results demonstrate that somatic cell nuclear transfer using transgenic skin cells as karyoplast donors is an alternative method to produce a herd of transgenic female dairy goats. Table 1. Caprine transgenic herd development by nuclear transfer