Isolation of Tanichthys albonubes beta actin gene and production of transgenic Tanichthys albonubes.
A beta actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5'UTR and a 564-bp 3'UTR. Genomic DNA containing the transcription region and 5'-flanking region was cloned based on the beta actin cDNA by Genome walker. A 3,000-bp beta actin gene promoter was then produced by PCR according to the sequences of the 5'-flanking region and the first intron. This promoter consisted of a 1,800-bp 5'-flanking region, and a 1,200-bp 5'-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5'-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The beta actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs. Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.
Yu
Er-meng
EM
College of Aqua-life Science and Technology, Shanghai Ocean University, Shanghai 200090, China.
Ye
Xing
X
Wang
Hai-ying
HY
Bai
Jun-jie
JJ
Xia
Shi-ling
SL
Lao
Hai-hua
HH
Jian
Qing
Q
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000199:Actins / Q000235:genetics*; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions; D003530:Cyprinidae / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D029941:Fish Proteins / Q000235:genetics*; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D000199:Actins; D022801:Complementarity Determining Regions; D017931:DNA Primers; D018076:DNA, Complementary; D029941:Fish Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C413663:fluorescent protein 583; D013006:Growth Hormone
Nat Protoc. 2006;1(3):1133-9
17406394
Mol Cell Biol. 1990 Jul;10(7):3432-40
2355913
Mol Mar Biol Biotechnol. 1994 Aug;3(4):192-9
8000477
J Cell Biol. 1986 Apr;102(4):1485-93
3007534
Transgenic Res. 2001 Aug;10(4):353-62
11592714
Mol Mar Biol Biotechnol. 1998 Sep;7(3):173-80
9701611
J Biol Chem. 1989 Jun 5;264(16):9539-46
2722849
Proc Natl Acad Sci U S A. 1988 Mar;85(5):1389-93
3422740
Biochim Biophys Acta. 2003 Jan 3;1625(1):11-8
12527420
Yi Chuan Xue Bao. 2006 Feb;33(2):133-40
16529297
Nucleic Acids Res. 1989 Jul 25;17(14):5850
2762162
Mar Biotechnol (NY). 2003 May-Jun;5(3):244-52
14502396
Nucleic Acids Res. 1984 Feb 10;12(3):1687-96
6322116
Fish Physiol Biochem. 2005 Apr;31(2-3):281-7
20035471
J Mol Biol. 1978 Dec 25;126(4):783-802
745245
Transgenic Res. 1991 Dec;1(1):31-7
1844573
Philos Trans R Soc Lond B Biol Sci. 1993 Feb 27;339(1288):225-32
8097052
Proc Natl Acad Sci U S A. 1984 May;81(10):2980-4
6328484
J Mol Biol. 1996 Jun 21;259(4):655-65
8683572
Science. 1984 Jun 29;224(4656):1436-8
6729461
Transgenic Res. 1998 Sep;7(5):357-69
9859224
Dev Biol. 1997 Dec 15;192(2):289-99
9441668