Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter.
The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.
Hwang
Gyu-Lin
GL
Division of Cell Sciences, School of Biological Sciences, University of Southampton, UK.
Azizur Rahman
M
M
Abdul Razak
Shaharudin
S
Sohm
Frédéric
F
Farahmand
Hamid
H
Smith
Alan
A
Brooks
Carly
C
Maclean
Norman
N
D000199:Actins / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D002347:Carps / Q000196:embryology / Q000235:genetics*; D008845:Microinjections; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D017210:Tilapia / Q000196:embryology / Q000235:genetics*; D019076:Transgenes
D000199:Actins