High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence.
In our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine beta-casein promoter. The expression level of the protein in milk of 7 mice ranged from 1 to 200 microg/ml; 1 to 34 microg/ml in 6 mice and 200 microg/ml in 1 mouse. With the aim of inducing higher expression of the protein, we constructed an expression cassette comprised of 10 kb of the bovine beta-casein gene promoter and the hLF genomic sequence in place of the cDNA. The hLF genomic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine beta-casein promoter. In total, 8 transgenic mice were generated from 31 mice (transgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hLF expression cassette. Mammary-specific expression of the transgene was addressed by performing Northern hybridization of the total RNAs from various tissues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native protein. The amount of the protein in milk of 5 mice ranged from 60 to 6,600 microg/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500 microg/ml. These data suggest that the genomic lactoferrin sequence represents a valuable element for the efficient expression of the protein in milk of transgenic animals.
Kim
S J
SJ
Animal Molecular Physiology Research Unit Korea Research Institute of Bioscience and Biotechnology, Taejon, 305-333, Korea.
Sohn
B H
BH
Jeong
S
S
Pak
K W
KW
Park
J S
JS
Park
I Y
IY
Lee
T H
TH
Choi
Y H
YH
Lee
C S
CS
Han
Y M
YM
Yu
D Y
DY
Lee
K K
KK
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002364:Caseins / Q000235:genetics; D002417:Cattle; D018076:DNA, Complementary / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000096:biosynthesis* / Q000235:genetics*; D008297:Male; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000737:chemistry*; D008957:Models, Genetic; D009928:Organ Specificity; D011401:Promoter Regions, Genetic
D002364:Caseins; D018076:DNA, Complementary; D007781:Lactoferrin