gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
pUASTB Drosophila Gene Expression Vector
Our pUASTB vector system is highly effective for generating transgenic flies and controlling transgene expression. This system has the capability to utilize either the Drosophila P-element transposon system (like pUAST) or the bacteriophage φC31 integration system (like pUASTattB), and incorporates a strong Gal4-inducible promoter to regulate transgene expression. Thus, the pUASTB vector system can be used for either P transposon-mediated or φC31 integrase-mediated insertion of your gene of interest. In either case, the user selected gene of interest is cloned into pUASTB in a region bracketed by two P-element terminal repeats and near an attB recombination site.
To utilize P transposon-mediated insertion, the pUASTB plasmid and a P transposase-expressing helper plasmid are co-introduced into host cells or embryos. As a result, the transposase produced from the helper plasmid recognizes the two P-element terminal repeats on the pUASTB plasmid, and inserts the flanked region including the two P-element terminal repeats into the host genome. P transposase-mediated insertion occurs without any significant bias with respect to insertion site sequence.
To utilize φC31 integrase-mediated insertion, the pUASTB plasmid and a φC31 integrase-expressing helper plasmid are co-introduced into host cells or embryos containing attP landing sites. The φC31 integrase mediates irreversible recombination between attB and attP sites, resulting in the linearization and integration of the pUASTB vector into the host genome.
The mini white gene on the pUASTB vector encodes for eye color and acts as a marker for the identification of transgenic flies which have undergone successful genetic recombination of the transgene.
In the pUASTB system, your gene of interest is cloned downstream of an engineered, inducible promoter consisting of five tandemly arrayed GAL4 binding sites (5xUAS) and the hsp70 TATA box promoter. This GAL4/UAS system is designed to direct selective, GAL4-dependent expression of the gene of interest. The GAL4 protein activates gene transcription upon binding to the UAS sites on the pUASTB plasmid. Therefore, in the absence of GAL4 expression the gene of interest remains silent, but introduction of GAL4 by crossing to a GAL4-expressing Drosophila line, results in transcriptional activation of the gene of interest in GAL4-expressing cells.
For further information about this vector system, please refer to the papers below.
|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|
Our pUASTB Drosophila gene expression vectors are designed to achieve either P transposase-mediated or φC31 integrase-mediated genomic insertion and selective, GAL4-dependent expression of a gene of interest. Our vectors are optimized for high copy number replication in E. coli and high-efficiency transgenesis of Drosophila lines.
Site-specific insertion: φC31-based insertion is site-specific, generally occurring only at attP sites, and not elsewhere in the genome. This reduces the risk of disrupting endogenous genes or having insertion site position effects on transgene expression.
High-level expression: The 5×UAS/mini_Hsp70 promoter can drive strong expression of the gene of interest in its induced state.
Selective expression: In the absence of GAL4, transcription of the gene of interest should be very low or silent, while in the presence of GAL4, high level of gene transcription is achieved.
High efficiency: Achieving germ-line transgenesis using φC31 integrase vectors is more efficient than P-element vector systems such as pUAST.
Random genomic insertion: If P transposase-mediated insertion of pUASTB is used, random integration of P-elements can make it difficult to map genomic insertion sites, and genomic position can affect transgene expression. Additionally, transgene insertion into genes or regulatory elements within the genome can affect endogenous genes.
Potentially leaky expression: In some cases, low-level expression of the gene of interest can occur in the absence of GAL4.
Technical complexity: The generation of transgenic Drosophila requires embryonic injection and fly husbandry, which can be technically difficult.
Requires attP insertion sites: The use of φC31 integrase-mediated insertion of pUASTB requires the use of specialized host lines carrying attP “landing sites” in their genome.
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. ØC31 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.