37 EFFECT OF CELL MANIPULATION FOR PRODUCTION OF TRANSGENIC CELL LINES ON GOAT CLONING EFFICIENCY

Abstract

The generation of transgenic cell lines through standard cell transfection/antibiotic selection procedures may have a negative effect on cell viability, which in turn may compromise SCNT cloning efficiency. The aim of this study was to evaluate goat cloning efficiency by using transfected and nontransfected and transgenic and nontransgenic somatic cells as nucleus donors. Skin fibroblast cells from 1 adult doe were subjected to transfection by electroporation with the pBC1-hGCase-Neo transgene cassette containing the human glucocerebrosidase gene sequence (hGCase), following antibiotic cell colony selection. Four distinct syngeneic donor cell types were used for cloning: (a) wild type (nontransfected, nontransgenic) control cells (C1) at low passage (P3), (b) transfected negative control (transfected, nontransgenic) cells (CT) at high passage (P8), and (c) 2 lines of transfected, transgenic cells (CA, CB) at high passages (P8 through P10). Donor cell cycles were synchronized by high confluence (<95%) and 24-h serum starvation. Cloning procedures were performed by standard micromanipulation procedures. Following membrane fusion after a 1.25 kV cm^–1 DC pulse for 45 µs, reconstructed structures were incubated in cytochalasin B for 1 h, and then activated in ionomycin/6-DMAP. After 12 h of IVC in G-1^TM medium (Vitrolife, Englewood, CO, USA), 1-cell stage cloned embryos were surgically transferred into the oviduct of synchronous recipient females. To ascertain herd fertility and health and adequate procedures for embryo manipulation, synchronization protocols, and surgical interventions, groups of control females were subjected to cervical AI or surgical transfer of in vivo-produced 1-cell stage goat embryos (ET). Pregnancy diagnosis was performed by ultrasonography on Day 23, with weekly examinations until term. Data were analysed by the χ² test (P < 0.05), and are presented in Table 1. The transfection process and passage number did not appear to affect development, as no differences in pregnancy rates were observed between cloned groups, although results with control cells (C1 and CT) and with CA and CB lines were similar to and lower than the AI and ET groups, respectively. Loss rate after cloning was high (88.8%), which may be due to faulty reprogramming, as other procedural and biological variables involved in the cloning process were endorsed by pregnancy rates and term viable pregnancies observed in the AI and ET groups. Cloning using CA donor cells at P9 resulted in two liveborn kids, with one dying soon after birth. Both animals were confirmed by molecular analyses as hGCase transgenic clones.

Table 1.  Overall efficiency after AI, superovulation and embryo transfer (ET) or cloning by nuclear transfer (NT) using control (C1), sham-transfected (CT), and 2 transgenic (CA, CB) syngeneic fibroblast cells lines in goats

This research was funded by FINEP.