Improved Production of Genetically Modified Fetuses with Homogeneous Transgene Expression After Transgene Integration Site Analysis and Recloning in Cattle
| Autor: | Mario Binelli, Tiago Henrique Câmara de Bem, Moysés dos Santos Miranda, Flávia Thomaz Verechia Pereira, Bryan E. Strauss, Marcio C. Bajgelman, Fabiana Fernandes Bressan, Felipe Perecin, Elisa Maria de Sousa Russo-Carbolante, Daiani Cristina Cilão Alves, José Eduardo Krieger, Flávio Vieira Meirelles |
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| Rok vydání: | 2011 |
| Předmět: |
Male
Nuclear Transfer Techniques Offspring Cloning Organism Transgene Biology Animals Genetically Modified Fetus BOVINOS Pregnancy medicine Animals Transgenes Blastocyst Gene Cells Cultured Cloning Embryo Cell Biology Fibroblasts Molecular biology Genetically modified organism medicine.anatomical_structure Cattle Female Developmental Biology Biotechnology |
| Zdroj: | Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 2152-4998 2152-4971 |
| DOI: | 10.1089/cell.2010.0022 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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