Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts

The efficiency of nuclear transfer (NIT) using two primary cultures of feta l fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence i s placed under the control of the bovine as alpha (s1)-casein promoter (TFF 1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgen ic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos rec onstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage a t a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were si gnificantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear do nors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NIT embryos der ived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majorit y of pregnancies (8/11; 73%) was lost in the first and second trimesters of gestation, whereas 4/11 (36%) pregnancies with FF2 embryos were lost durin g the full period of in vivo development. Of 11 FF2 and 6 TFF2 born calves (25 and 13% of transferred embryos, respectively), 6 and 3 survived includi ng one oversized FF2 calf. After transfer of TRFF1 and TRCF1 NT embryos to recipients, initial pregnancy rate was as a tendency higher in the TRFF1 (4 9%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancie s and all TRCF1 pregnancies were lost in the first and second trimester. A high proportion of TRFF1 calves (5/8) showed increased body weights, and on ly two calves which were also large survived. These findings demonstrate th at (i) extended culture associated with transfection and selection procedur es may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning. (C) 2001 Wiley-Liss, Inc.