Artículos de revistas
Improved Production of Genetically Modified Fetuses with Homogeneous Transgene Expression After Transgene Integration Site Analysis and Recloning in Cattle
Fecha
2011Registro en:
CELLULAR REPROGRAMMING, v.13, n.1, p.29-36, 2011
2152-4971
10.1089/cell.2010.0022
Autor
BRESSAN, Fabiana Fernandes
MIRANDA, Moyses dos Santos
PERECIN, Felipe
BEM, Tiago Henrique De
PEREIRA, Flavia Thomaz Verechia
RUSSO-CARBOLANTE, Elisa Maria
ALVES, Daiani
STRAUSS, Bryan
BAJGELMAN, Marcio
KRIEGER, Jose Eduardo
BINELLI, Mario
MEIRELLES, Flavio Vieira
Institución
Resumen
Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.