Molecular analysis of the first intron in the bovine myostatin gene.
To study the mechanism of transcription and expression of the myostatin gene, we cloned and analyzed the sequence of the bovine myostatin gene promoter and first intron from Qinchuan and Red Angus cattle, then constructed eukaryotic expression vectors encoding the GFP vector by replacing the CMV promoter with the bovine myostatin promoter using PCR method, thereby obtaining an expression vector coding GFP report gene with first intron (identified as pEGFP-MSTNPro-intron1). By transfecting C2C12 cells with the vectors, we then compared the effect on GFP gene expression of the promoter and normal first intron of Qinchuan and Red Angus cattle with that from the promoter and a Qinchuan allele with a 16 base pair insertion. After 48 h incubation, fluorescent indices (FIs), which indicate the expression rate and intensity of gene GFP expression, were analyzed by flow cytometry (FCM). Results showed that Qinchuan sequence homology of promoter was 99% with Red Angus, that Qinchuan first intron sequence homology was 99.51% with Red Angus and that first intron homologies of Qinchuan and Red Angus were 99.08 and 99.02%, respectively, with Accession No.AF320998 in GenBank. Expression of the GFP gene did not differ significantly between preparations using the Qinchuan versus Red Angus promoter. Preparations with a construct that included the first intron had higher GFP gene expression in C2C12 cells than those whose construct lacked the first intron (P < 0.05 or P < 0.01). However, there was no significant difference (P > 0.05) in gene expression between normal first intron and 16 bp insertion first intron (+16 bp) preparations.
Key Laboratory of Animal Reproductive Endocrinology and Embryo Engineering, Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Yulong
Y
He
Yuehong
Y
Wu
Zhigang
Z
Lan
Yonggang
Y
Liu
Yong
Y
Zhang
D000818:Animals; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D002460:Cell Line; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D055435:Myostatin / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D055435:Myostatin; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins