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Construction and Expression Analysis of Knock-in Vector for EGFP Expression in the Porcine $\beta$-Casein Gene Locus  

Lee, Sang-Mi (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University)
Kim, Hey-Min (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University)
Moon, Seung-Ju (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University)
Kang, Man-Jong (Department of Animal Science and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonnam National University)
Publication Information
Reproductive and Developmental Biology / v.32, no.3, 2008 , pp. 205-209 More about this Journal
Abstract
This study was carried out to develop knock-in vector for EGFP (enhanced green fluorescent protein) expression in porcine $\beta$-casein locus. For construction of knock-in vector using porcine $\beta$-casein gene, we cloned the $\beta$-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 ($neo^{r}$) 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.
Keywords
$\beta$-Casein gene; Knock-in vector; HC11 cells; Gene targeting;
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