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The Chinese Academy of Agricultural Sciences (CAAS) is a national, integrative agricultural scientific research organization with responsibility for carrying out both basic and applied research, as well as research into new technologies impacting agriculture. CAAS is dedicated to overcoming a broad range of challenges impacting agricultural development and support of the local rural economy. Importantly, CAAS promotes sustainable agriculture within and outside China, extending its reach through technology exchange and cooperative research agree [...]

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Journal Article

Journal Article

Construction and functional analysis of a pig myostatin gene targeting vector containing double-selection markers  [2012]

Wang Xiangpeng, Northwest Agriculture and Forestry University, Yangling (China); Xiao Yihong, Shandong Agricultural University, Taian (China), College of Veterinary Medicine; Liu Yuanyuan, Northwest Agriculture and Forestry University, Yangling (China);

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A pig myostatin (MSTN) gene targeting vector containing double-selection markers was constructed based on the positive-negative selection strategy. The MSTN gene homologous long arm and short arm were cloned in the genome of pig fetus fibroblasts cells by PCR method. The positive selection cassette puromycin(Puro) and enhanced green fluorescent protein(EGFP) genes and the negative selection cassette thymidine kinase(TK) gene were cloned by PCR and Overlap PCR. A set of cassettes and plasmids was used for creating the targeting vector. A positive selection marker cassette (PGK-Puro-EGFP) including the eukaryotic promoter to permit consecutive selection for recombination and allow positive selection of puromycin resistant transfectants in mammalian cells was flanked by two Frt sequences. EF1?-herpes simplex virus thymidine kinase (TK) minigene was used as the negative selection marker. The homologous long arm with a length of 5.7 kb amplified from pig embryonic fibroblast cell genomic DNA was placed before the first Frt sequence. The homologous short arm with a length of 1.9 kb was cloned between the positive and negative selection cassettes. The plasmid was transfected into PK15 cells with X-tremeGene HP DNA transfection reagent to evaluate the positive and negative selection markers function. The homologous long arm and short arm together with the Puro and EGFP and TK gene were successfully cloned. The presence of the correct inserts in the targeting vecto
r with the full length of 14 kb was verified by DNA sequencing. Meanwhile, the PK15 cells conferred resistance to puromycin (Puro) and ganciclovir (GAC), which suggested the positive and negative selection markers could expressed in the cells. A pig myostatin (MSTN) gene targeting vector containing double-selection markers was constructed.
From the journal
Journal of Northwest Agriculture and Forestry University
ISSN : 1671-9387

Bibliographic information

Language:
Chinese
Type:
Journal Article
In AGRIS since:
2013
Volume:
40
Issue:
8
Start Page:
47
End Page:
53
All titles:
"Construction and functional analysis of a pig myostatin gene targeting vector containing double-selection markers"@eng
"?MSTN????????????????????"@chi
Other:
"Resume(Zh,En)"
"15 ref."
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Bibliographic information

Language:
Chinese
Type:
Journal Article
In AGRIS since:
2013
Volume:
40
Issue:
8
Start Page:
47
End Page:
53
All titles:
"Construction and functional analysis of a pig myostatin gene targeting vector containing double-selection markers"@eng
"?MSTN????????????????????"@chi
Other:
"Resume(Zh,En)"
"15 ref."