In vitro and ex vivo green fluorescent protein expression in alveolar mammary epithelial cells and mammary glands driven by the distal 5'-regulative sequence and intron 1 of the goat beta-casein gene.
The 5'-regulative sequence and intron 1 of the goat beta-casein gene from -4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5'-regulative sequence and intron 1 of the 6.2 kb goat beta-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.
Wu
Hsi-Tien
HT
Institute of Animal Science, National Chung-Hsing University, Taichung, Taiwan.
Lin
Chich-Sheng
CS
Huang
Mu-Chiou
MC
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D019677:Artificial Gene Fusion; D002364:Caseins / Q000235:genetics*; D002460:Cell Line; D004847:Epithelial Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006041:Goats; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007438:Introns; D007774:Lactation / Q000235:genetics; D008321:Mammary Glands, Animal / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008894:Milk Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011388:Prolactin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014162:Transfection; D019076:Transgenes
D002364:Caseins; D008894:Milk Proteins; D049452:Green Fluorescent Proteins; D011388:Prolactin