Document Detail


Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure.
MedLine Citation:
PMID:  22898014     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
The objective was to use dual markers to accurately select genetically modified donor cells and ensure that the resulting somatic cell nuclear transfer kids born were transgenic. Fetal fibroblast cells were transfected with dual marking gene vector (pCNLF-ng) that contained the red-shifted variant of the jellyfish green fluorescent protein (LGFP) and neomycin resistance (Neo) markers. Cell clones that were G418-resistant and polymerase chain reaction-positive were subcultured for several passages; individual cells of the clones were examined with fluorescence microscopy to confirm transgenic integration. Clones in which every cell had bright green fluorescence were used as donor cells for nuclear transfer. In total, 86.7% (26/30) cell clones were confirmed to have transgenic integration of the markers by polymerase chain reaction, 76.7% (23/30) exhibited fluorescence, but only 40% (12/30) of these fluorescent cell clones had fluorescence in all cell populations. Moreover, through several cell passages, only 20% (6/30) of the cell clones exhibited stable LGFP expression. Seven transgenic cloned offspring were produced from these cells by nuclear transfer. Overall, the reconstructed embryo fusion rate was 76.6%, pregnancy rates at 35 and 60 days were 39.1% and 21.7%, respectively, and the offspring birth rate was 1.4%. There were no significant differences between nuclear transfer with dual versus a single (Neo) marker (overall, 73.8% embryo fusion rate, 53.8% and 26.9% pregnancy rates, and 1.9% birth rate with five offspring). In conclusion, the use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats. Furthermore, the LGFP/Neo markers had no adverse effects on the efficiency of somatic cell nuclear transfer.
Authors:
Li-You An; Yu-Guo Yuan; Bao-Li Yu; Ting-Jia Yang; Yong Cheng
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-8-13
Journal Detail:
Title:  Theriogenology     Volume:  -     ISSN:  1879-3231     ISO Abbreviation:  Theriogenology     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-8-17     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0421510     Medline TA:  Theriogenology     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012 Elsevier Inc. All rights reserved.
Affiliation:
College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China, Engineering Research Centre for transgenic Animal Pharmaceutics in Jiangsu Province, Yangzhou, Jiangsu, China.
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