The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice.
Various combinations of promoters, introns and transcription terminators were used to drive the expression of bovine growth hormone (bGH) cDNA in different cell types. In constructs containing the human cytomegalovirus (hCMV) promoter and the SV40 late genes terminator, the intron from SV40 genes (VP1) was much more efficient, than the intron from the early genes (t). The synthetic intron SIS generated by the association of an adenovirus splice donor and an immunoglobulin G splice acceptor showed the highest activity. The respective potency of these introns was similar in several mammalian (CHO, HC11 and COS) and fish (TO2 and EPC) cells. The rabbit whey acidic protein (WAP) gene promoter was highly efficient to drive the expression of bGH gene in the HC11 mammary cell lines. In contrast, the bGH cDNA under the control of the same promoter was much less efficiently expressed when the SV40 VP1 intron and transcription terminator were used. The rabbit WAP gene and the human GH gene terminators did not or only moderately enhanced the expression of the construct WAP bGH cDNA. Introduction of a promoter sequence from the mouse mammary tumor virus (MMTV) LTR in the VP1 intron increased very significantly the expression of the WAP bGH cDNA. Although several of these vectors showed high potency when expressed stably in HC11 cells, all of them were only moderately efficient in transgenic mice. These data indicate that the VP1 and the SIS introns may be used to express foreign cDNAs with good efficiency in different cell types. The addition of an enhancer within an intron may still reinforce its efficiency. However, transfection experiments, even when stable expression is carried out, are poorly predictive of the potential efficiency of a vector in transgenic animals.
Petitclerc
D
D
Agriculture et Agro-Alimentaire Canada, Est Lennoxville, Quebec.
Attal
J
J
Théron
M C
MC
Bearzotti
M
M
Bolifraud
P
P
Kann
G
G
Stinnakre
M G
MG
Pointu
H
H
Puissant
C
C
Houdebine
L M
LM
D000818:Animals; D001709:Biotechnology; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002417:Cattle; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D007438:Introns; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008894:Milk Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D013539:Simian virus 40 / Q000235:genetics; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D036022:Capsid Proteins; D018076:DNA, Complementary; D008894:Milk Proteins; D012333:RNA, Messenger; C024471:whey acidic proteins; D013006:Growth Hormone