果蝇基因表达双系统载体pUASTB

我们的pUAST果蝇基因表达载体可有效实现P转座酶或φC31整合酶介导的基因插入或目的基因GAL4依赖性表达。我们的载体在大肠杆菌中具有高拷贝，可用于高效产生转基因果蝇。

 

参考文献	主题
Development. 118:401 (1993)	Development and use of the pUAST inducible promoter system
Methods Mol Biol. 420:61 (2008)	The use of P-element transposons to generate transgenic flies
Proc Natl Acad Sci U S A. 97:5995 (2000) Proc Natl Acad Sci U S A. 95:5505 (1998)	Description of the φC31 integrase system
Proc Natl Acad Sci U S A. 104:3312 (2007)	Use of pUASTattB and germ-line-specific φC31 integrase to construct transgenic Drosophila
Genetics. 166:1775 (2004)	Use of pUASTB to construct transgenic Drosophila

特定位点插入：基于φC31的插入具有位点特异性，通常仅发生在attP位点，降低了破坏内源基因或插入位点不当影响转基因表达的风险。

高水平表达：5×UAS/mini_Hsp70启动子可以在诱导状态下使目的基因高水平表达。

选择性表达: 不存在GAL4的情况下，目的基因的转录水平很低或没有；在GAL4的存在下，则能实现高水平的基因转录。

高效：使用φC31整合酶实现种系转基因比基于P元件的载体系统如pUAST更有效。


载体关键原件


 

P-element 3’ end: Right terminal repeat, or 3' terminal repeat, of the P element. When a DNA sequence is flanked by the 3’ and 5’ P-element terminal repeats, the P transposase can recognize them and insert the flanked region into the host genome.

5×UAS/mini_Hsp70: The Drosophila melanogaster heat shock protein 70 (Hsp70) minimal promoter fused with five tandem galactose upstream activating sequences (5×UAS). This is a strong promoter, tightly inducible by GAL4.

Kozak: Drosophila Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

SV40 terminator: Simian virus 40 transcriptional terminator. Contains the SV40 small T intron and the SV40 early polyadenylation signal.

attB site: The bacterial attachment site, attB, recognized by the bacteriophage φC31 serine integrase. φC31 integrase can catalyze site-specific integration of attB-containing plasmids into attP-containing docking or landing sites that have been introduced into host genomes.

mini-white: A variant of the Drosophila white gene. The mini-white gene is a dominant marker for adult fruit fly eye color, which can be used as a reporter to identify transgenic events in a white mutant background.

P-element 5’ end: Left terminal repeat, or 5' terminal repeat, of the P element. When a DNA sequence is flanked by the 3’ and 5’ P-element terminal repeats, the P transposase can recognize them and insert the flanked region into the host genome.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.