In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype

被引:77
作者
Arat, S
Gibbons, J
Rzucidlo, SJ
Respess, DS
Tumlin, M
Stice, SL
机构
[1] Univ Georgia, Dept Anim & Dairy Sci, Athens, GA 30602 USA
[2] TUBITAK, Res Inst Genet Engn & Biotechnol, TR-41470 Gebze, Kocaeli, Turkey
[3] Prolinia Inc, Athens, GA 30602 USA
关键词
assisted reproductive technology; developmental biology; embryo;
D O I
10.1095/biolreprod66.6.1768
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 105 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 NIT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.
引用
收藏
页码:1768 / 1774
页数:7
相关论文
共 61 条
[1]   Remodeling of donor nuclei, DNA-synthesis, and ploidy of bovine cumulus cell nuclear transfer embryos:: Effect of activation protocol [J].
Alberio, R ;
Brero, A ;
Motlík, J ;
Cremer, T ;
Wolf, E ;
Zakhartchenko, V .
MOLECULAR REPRODUCTION AND DEVELOPMENT, 2001, 59 (04) :371-379
[2]   Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes [J].
Arat, S ;
Rzucidlo, SJ ;
Gibbons, J ;
Miyoshi, K ;
Stice, SL .
MOLECULAR REPRODUCTION AND DEVELOPMENT, 2001, 60 (01) :20-26
[3]  
Arat S, 2001, BIOL REPROD, V64, P173
[4]   Production of goats by somatic cell nuclear transfer [J].
Baguisi, A ;
Behboodi, E ;
Melican, DT ;
Pollock, JS ;
Destrempes, MM ;
Cammuso, C ;
Williams, JL ;
Nims, SD ;
Porter, CA ;
Midura, P ;
Palacios, MJ ;
Ayres, SL ;
Denniston, RS ;
Hayes, ML ;
Ziomek, CA ;
Meade, HM ;
Godke, RA ;
Gavin, WG ;
Overström, EW ;
Echelard, Y .
NATURE BIOTECHNOLOGY, 1999, 17 (05) :456-461
[5]   Bypass of senescence after disruption of p21(CIP1/WAF1) gene in normal diploid human fibroblasts [J].
Brown, JP ;
Wei, WY ;
Sedivy, JM .
SCIENCE, 1997, 277 (5327) :831-834
[6]   GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION [J].
CHALFIE, M ;
TU, Y ;
EUSKIRCHEN, G ;
WARD, WW ;
PRASHER, DC .
SCIENCE, 1994, 263 (5148) :802-805
[7]  
Chan AWS, 1999, MOL REPROD DEV, V52, P406, DOI 10.1002/(SICI)1098-2795(199904)52:4&lt
[8]  
406::AID-MRD9&gt
[9]  
3.0.CO
[10]  
2-P