[Cloning of 5', 3' flanking sequence of ovine BLG and regulating the expression of GFP in mammary gland cell line].
5' and 3' flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently. By partially sequencing, the sequences of BLG 5' and 3' flanking were the same as that of publication completely. The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2 kb 5' flanking with 2.1 kb 3' flanking. In order to assess the efficiency of BLG regulatory elements, green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47). Through observation under UV microscope and detection by fluorometer, it is demonstrated that the GFP has been successfully expressed in TD47 cell line. By virtue of direct observation and quantitative analysis, the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.
Liu
Ming-Jun
MJ
Xinjiang Academy of Animal Science, Urumqi 830000, China.
Li
Wen-Rong
WR
Wu
Jian
J
Huang
Jun-Cheng
JC
Guo
Zhi-Qin
ZQ
Qu
Xin-Yong
XY
Paul
Kroon
K
D024509:3' Flanking Region / Q000235:genetics*; D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001940:Breast / Q000166:cytology; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007782:Lactoglobulins / Q000096:biosynthesis / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D012756:Sheep
D007782:Lactoglobulins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins