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

Abstract

The efficiency of nuclear transfer (NT) using two primary cultures of fetal fibroblasts (FF1 and FF2) was compared vs. the same cultures transfected with an expression vector in which the bovine prochymosin coding sequence is placed under the control of the bovine alpha(S1)-casein promoter (TFF1 and TFF2). In addition, fibroblasts of a cloned transgenic fetus (TRFF1) derived from TFF1 and ear skin fibroblasts of a 1-month-old cloned transgenic calf (TRCF1) derived from TRFF1 were used as nuclear donors. Embryos reconstructed from FF1 (44%) and FF2 (52%) developed to the blastocyst stage at a significantly (P < 0.05) higher rate than those derived from TFF1 (24%) and TFF2 (27%). The proportions of cleaved embryos and blastocysts were significantly (P < 0.05) higher with TRFF1 than with TRCF1 used as nuclear donors (75 vs. 66% and 33 vs. 16%, respectively). Transfer of NT embryos derived from FF2 and TFF2 to recipients resulted in similar pregnancy rates on day 30 (52 and 48%, respectively). However, with TFF2 embryos, the majority 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 during 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 including 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 (49%) than in the TRCF1 group (30%). The majority (14/17) of TRFF1 pregnancies 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 only two calves which were also large survived. These findings demonstrate that (i) extended culture associated with transfection and selection procedures may induce changes of donor cells which markedly decrease the efficiency of nuclear transfer and (ii) these changes are not reversed by recloning.

Effects of Recloning on the Efficiency of Production of .ALPHA.1,3-Galactosyltransferase Knockout Pigs

“…Iguma et al [25], Forsberg et al [26] and Zakhartchenko et al [27] reported that in the case of cattle, genetic modification of donor cells influences the number of cloned calves born. An increase in chromosomal aberration, such as mutation and deletion due to a long culturing period, is considered one of the causes.…”

Transgenic bovine as bioreactors: Challenges and perspectives

“…47 However, somatic cells have a limited life span in in vitro culture, and the decrease in developmental rates for modified cells has been attributed to the extended culture periods associated with the transfection and selection procedures before SCNT. 48 Studies using different approaches aimed at increasing the efficiency for the generation of viable clones have been performed. The methods investigated include recloning, autologous SCNT, impeding Xist expression from the active X chromosome, treatment with trichostatin A after SCNT and histone deacetylase inhibitors, among others.…”

Transgenesis for the study and the control of lactation

“…The integrated gene can be examined before proceeding to nuclear transfer. This method is without contest more attractive than microinjection for sheep [35], goats [19,32] and cows [2,43].…”