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

Abstract

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