Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.
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.
Department of Basic Sciences, FZEA, University of São Paulo , Pirassununga/São Paulo, Brazil. fabianabressan@usp.br
Fabiana Fernandes
FF
Bressan
Moyses
M
Dos Santos Miranda
Felipe
F
Perecin
Tiago Henrique
TH
De Bem
Flavia Thomaz Verechia
FT
Pereira
Elisa Maria
EM
Russo-Carbolante
Daiani
D
Alves
Bryan
B
Strauss
Marcio
M
Bajgelman
José Eduardo
JE
Krieger
Mario
M
Binelli
Flavio Vieira
FV
Meirelles
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000662:veterinary*; D005260:Female; D005333:Fetus / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D008297:Male; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D019076:Transgenes