73 EFFECT OF CALF RECLONING ON EMBRYO AND FETAL SURVIVAL

Abstract

In a large-scale cloning program destined to produce transgenic animals, it is very important to incorporate well-characterized transgene integration and gene expression. However, after non-homologous transfection, a wide variety of transgene copies are introduced, and these occur in different chromosome locations. Recloning a selected first-generation transgenic calf offers the opportunity to increase the homogeneity among transgenic animals. Calf recloning was performed in an experiment in which the survival rate was evaluated after a second round of cloning from transgenic umbilical cord and ear calf fibroblasts. The original genetically modified fetal cell line that produced the clones was used as control. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg mL⁻¹ bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg mL⁻¹) under UV light (<6 s). A fetal fibroblast cell line was initially established from a 75-day old Jersey female fetus. Genetically modified cells were isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. Following nuclear transfer with these transgenic cells, new cell lines were isolated from umbilical cord and ear fibroblasts obtained from one of the cloned-transgenic calves so-produced. Donor cells from all three sources were used for nuclear transfer at G0/G1 cell cycle stages and were fused to enucleated oocytes by an electrical pulse. After 3 h, activation was induced by incubation in TL-HEPES with 5 μM ionomycin for 4 min and then 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL- HEPES and cultured in SOF medium with an atmosphere of 5% CO₂ + 5% O₂ + 90% N₂. Development to blastocyst stage (Days 7 to 9) was recorded. One or two blastocysts were transferred non-surgically per recipient cow, and pregnancies at 30 days or 60 days were determined by ultrasonography. All data were analyzed by chi-square test. Seven births were obtained from the original fetal cell line, one birth was obtained from recloned umbilical cord and four pregnancies are in the last third of gestation from recloned ear fibroblasts. Development to blastocyst stage was significantly different between transfected fetal fibroblast and both recloned treatment groups. Differences were observed in pregnancy rates between blastocysts generated by the different sources of donor cells. In spite of the lower blastocyst production, our results suggest that recloning provides an additional method to obtain transgenic animals, and that fibroblasts from umbilical cord could give better results for recloning than those obtained from young calf ear.

Effect of different sources of donor cells for calf recloning on embryo and fetal survival