在pUASTB系统中,目的基因将被克隆到合成诱导型启动子的下游,该启动子由5个串联排列的GAL4结合位点(5xUAS)和hsp70 TATA box组成。目的基因依赖于GAL4的表达,GAL4蛋白在与pUAST质粒上的UAS位点结合后会激活目的基因的转录。因此,在没有GAL4表达的情况下,目的基因保持沉默,但是通过与表达GAL4的果蝇系杂交后,在GAL4表达的细胞中,目的基因会被转录激活。
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.