Bressan Fabiana Fernandes, Dos Santos Miranda Moyses, Perecin Felipe, De Bem Tiago Henrique, Pereira Flavia Thomaz Verechia, Russo-Carbolante Elisa Maria, Alves Daiani, Strauss Bryan, Bajgelman Marcio, Krieger José Eduardo, Binelli Mario, Meirelles Flavio Vieira
Department of Basic Sciences, FZEA, University of São Paulo , Pirassununga/São Paulo, Brazil.
Cell Reprogram. 2011 Feb;13(1):29-36. doi: 10.1089/cell.2010.0022. Epub 2011 Jan 17.
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.
通过核移植（NT）进行动物克隆使得利用基因修饰的供体细胞生产转基因动物成为可能，并确保基因构建体存在于后代中。在克隆前鉴定供体细胞中的转基因插入位点可以避免由于位置效应而产生具有不良特征的动物。本文比较了用含有转基因随机整合的慢病毒介导的转基因成纤维细胞（随机整合组）或提交用于再克隆的具有已知转基因插入位点的核移植衍生转基因成纤维细胞重建的牛克隆胚胎的囊胚发育和建立妊娠的能力。在随机整合组中，通过荧光激活细胞分选（FACS）选择表达eGFP的牛胎儿成纤维细胞，并用作核移植的供体细胞。在再克隆组中，对来自转基因克隆胎儿的成纤维细胞系进行转基因插入特征分析，并进行再克隆。与随机整合组相比，再克隆组的囊胚产量更高（25.38%对14.42%），30天妊娠的百分比更高（14.29%对2.56%）。对每个克隆胚胎衍生的成纤维细胞中的相对eGFP表达分析显示，再克隆组中的表达更均匀。总之，在鉴定转基因整合位点后使用从转基因胎儿中回收的细胞系可以产生具有稳定转基因表达的细胞和胎儿，并且再克隆可以提高转基因动物的产量。
