Analysis of control elements for position-independent expression of human alpha-lactalbumin YAC.
A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high-expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human alpha-lactalbumin gene express the transgene in a position-independent manner. The 210 kb YAC was thought to have all the elements necessary for position-independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the alpha-lactalbumin gene had position-independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position-dependent expression. Therefore, all the elements necessary for position-independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the alpha-lactalbumin gene. Furthermore, we replaced the human alpha-lactalbumin promoter with the bovine alphaS1-casein promoter in the 210 kb YAC and produced transgenic rats. Position-dependent expression was observed. The elements required for position-independent expression of the bovine alphaS1-casein gene are different from those required for the human alpha-lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity.
Fujiwara
Y
Y
YS New Technology Institute Inc., Ishibashi-machi, Shimotsuga-gun, Tochigi, Japan.
Takahashi
R I
RI
Miwa
M
M
Kameda
M
M
Kodaira
K
K
Hirabayashi
M
M
Suzuki
T
T
Ueda
M
M
D000818:Animals; D030801:Animals, Genetically Modified; D002364:Caseins / Q000235:genetics; D002417:Cattle; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids; D005786:Gene Expression Regulation / Q000235:genetics*; D019382:Human Growth Hormone / Q000235:genetics; D006801:Humans; D007768:Lactalbumin / Q000235:genetics*; D019901:Locus Control Region / Q000235:genetics; D008894:Milk Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D002364:Caseins; D008894:Milk Proteins; D012333:RNA, Messenger; D019382:Human Growth Hormone; D007768:Lactalbumin