In Vitro Development of Bovine Nuclear Transfer Embryos from Transgenic Clonal Lines of Adult and Fetal Fibroblast Cells of the Same Genotype1

Abstract

This study examined bovine cloning strategies that may be used for gene targeting in animals of known phenotypic traits. Fibroblast cells derived from an adult and a fetus of the same genotype were transfected with a plasmid (pEGFP-N1) containing the enhanced green fluorescence protein and neomycin-resistant genes. After transfecting 2 x 10(5) cells, 49 adult and 35 fetal cell colonies were obtained. Green fluorescence expression was observed in 35 out of 49 (71.4%) adult clones and in 30 out of 35 (85.7%) fetal clones. Developmental rates to the blastocyst stage following nuclear transfer (NT) did not differ among nontransfected cell lines (adult, 20.0%; NT fetal, 18.3%), whereas developmental rates were significantly lower for adult and fetal cell lines expressing enhanced green fluorescent protein (EGFP; 11.3% and 6.4%, respectively, P < 0.05). However, there was no decrease in NT developmental rates (19.8%) when donor nuclei from EGFP-transfected cell lines not expressing EGFP but retaining neomycin-resistant gene expression were used as donor nuclei. NT embryos from adult and fetal cell lines had similar morphology, cell number, and ploidy. The results indicated that adult and NT fetal cells (identical genotype) can complete clonal propagation, including transfection and selection, and can be used to produce transgenic NT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.

Development Potential of Transgenic Somatic Cell Nuclear Transfer Embryos According to Various Factors of Donor Cell

“…This result suggested that ear fibroblasts affected more by transfection of foreign gene than cumulus cells with respect to blastocyst formation and the ability to tolerate the insult of transfection might be different between 2 types of cells. Although development rates of transgenic SCNT embryos derived from both of fibroblast were lower than those with cumulus cells, it was not lower than other reports [3,33]. In addition, GFP expression rates were evaluated according to embryo development.…”

“…These results suggest that an expression plasmid was suc- Model effect of the cell size on the number of embryos fused, cleaved, developed to the blastocysts and expressed, which was indicated as a P value, was 0. cessfully integrated into a chromosome of blastocysts and then GFP transfection system without establishing stable somatic cell lines can be used for the production of transgenic SCNT embryos. Previous reports showed that three types of somatic cells including fetal fibroblasts [15,32,46], granulosa cells [2] and ear fibroblasts [3] could be used for the production of transgenic bovine SCNT embryos with various efficacies. Based on our previous reports [13,21], three types of donor cells (cumulus cells, adult and fetal fibroblasts) were selected and used for the generation of transgenic SCNT embryos in this study.…”

“…Fetal cells have been used for transgenic cattle production because of their rapid growth and potential for many cell divisions before senescence in culture [5,15,28]. Among adult cells used for cloned animal production, only granulosa cells and ear fibroblasts have been utilized in transgenic SCNT [2,3]. Thus, the search for an ideal type of donor cells is an important step towards improving efficiency of bovine transgenic SCNT and subsequent production of transgenic cloned animals.…”

Evaluation of laser-assisted lentiviral transgenesis in bovine

“…Therefore numerous reports exist, where this research tool is used to obtain information about transgenesis, gene targeting and NT in bovine in vitro (Iguma et al, 2005, Cho et al, 2004, Arat et al, 2002, Wells and Powell, 2000, Rosochacki et al, 2003.…”

Produção de animais transgênicos por transferência nuclear como modelo de estudo biológico

“…O sucesso de desenvolvimento a blastocisto é normalmente atingido por 7 a 30%, aproximadamente, dos embriões reconstruídos por TN (ROH et al, 2000;CHO ET al., 2004;LEE et al, 2007). Já o sucesso do estabelecimento de prenhezes pode variar de 6 a 48%, (ZAKHARTCHENKO et al, 2001;ARAT et al, 2002;GONG et al, 2004;IGUMA et al, 2005 Todavia, como discutido acima, o tempo de cultivo pode não ser prejudicial à reprogramação nuclear destas células. É provável que outros fatores sejam responsáveis pela maior ou menor competência de reprogramação nuclear e sustentação do desenvolvimento embrionário.…”