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KMID : 1023620080320030205
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Reproductive and Developmental Biology
2008 Volume.32 No. 3 p.205 ~ p.209
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Construction and Expression Analysis of Knock-in Vector for EGFP Expression in the Porcine -Casein Gene Locus
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Lee Sang-Mi
Kim Hye-Min
Moon Seung-Ju
Kang Man-Jong
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Abstract
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This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine -casein locus. For construction of knock-in vector using porcine -casein gene, we cloned the -casein genome DNA from porcine fetal fibroblast cells, EGFP and SV40 polyA signal using PCR. The knock-in vectors consisted of a 5-kb fragment as the 5’ recombination arm and a 2.7-kb fragment as the 3’ recombination arm. We used the neomycin resistance gene () as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. To demonstrate EGFP expression from knock-in vector, we are transfected knock-in vector that has EGFP gene in murine mammary epithelial cell line HC11 cells with pSV2 neo plasmid. The EGFP expression was detected in HC11 cells transfected knock-in vector. This result demonstrates that this knock-in vector may be used for the development of knock-in transgenic pig.
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KEYWORD
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-Casein gene, Knock-in vector, HC11 cells, Gene targeting
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