Effect of donor cell transfection events on embryo and fetal survival in cloning.

SALAMONE DF; SANTOS C; BARAÑAO JL; BUSSMANN L; ARTUSO J; MUNAR CJ; BERRA G; CRISCUOLO M; MELO C

Kioto, Japón.

Congreso; Congreso anual de la IETS,; 2007

IETS

In a large-scale bovine cloning program intended to obtain transgenic animals, it is important to maximize the number of calves produced. The present experiment was designed to test the hypothesis that different transfection events of the same somatic cell line can affect embryo and/or fetal survival. A fetal cell line was established from a 75-day-old Jersey female fetus. It was used as control and was also transfected 3 times with the same protocol. They were named Transfection 1, 2, and 3. Genetically modified cells were produced and isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. 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). Donor cells from different treatments 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 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 blastocysts (Days 7 to 9) was recorded. Two blastocysts were transferred nonsurgically per recipient cow, and pregnancies at 30 days were determined by ultrasonography. All data were analyzed by chi-square test. In vitro development to blastocysts was similar in all treatment groups. One birth was obtained from the control. Four and 7 births were obtained from Transfections 1 and 3, respectively. Although Transfection 2 had good in vitro development, this treatment did not produce any pregnancy. This fact demonstrated that the transfection event provides an additional source of variability in obtaining live transgenic animals. Our results pointed out the necessity to monitor fetal survival by ultrasonography in order to detect as soon as possible any deficiencies in development introduced by transfection.