gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
pUASTattB Drosophila Gene Expression Vector
Our pUASTattB vector system is highly effective for generating transgenic flies and controlling transgene expression. This system utilizes the bacteriophage φC31 integrase-mediated recombination for efficient, targeted gene insertion, and incorporates a strong Gal4-inducible promoter to regulate transgene expression. The pUASTattB vector system is similar to the pUAST system, however it utilizes a φC31 integrase mediated recombination event rather than P-element based transposition for genomic insertion of your gene of interest.
The bacteriophage φC31 encodes an integrase that mediates efficient, sequence-specific recombination between phage attachment sites (called attP) and bacterial attachment sites (called attB). In contrast to transposon-based systems, such as P-element-mediated recombination, φC31-mediated insertion is irreversible. Integration of the pUASTattB into an attP site creates hybrid sites (called attL and attR), which are refractory to the φC31 integrase. Additionally, φC31-based insertion is site-specific, generally occurring only at attP sites, and not elsewhere in the genome. For this reason, the pUASTattB vector system is designed to be used with Drosophila lines carrying attP “landing sites” within their genome.
The pUASTattB system consists of two vectors, both engineered as E. coli plasmids. One vector referred to as the pUASTattB vector or the φC31 donor vector carries the attB site and the user’s gene of interest. The other vector referred to as the helper plasmid encodes for the φC31 integrase. When the pUASTattB and the φC31 helper plasmids are co-injected into cells containing attP landing sites, φC31 integrase-mediated recombination between attB and attP sites, results in the linearization and integration of the pUASTattB vector into the host genome. The mini white gene on the pUASTattB 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.
There are several possible methods for introduction of the φC31 integrase into Drosophila hosts. Target cells can be injected with φC31 integrase mRNA generated by in vitro transcription. Alternatively, a helper plasmid expressing φC31 integrase may be co-injected into the cells along with the pUASTattB vector. Additionally, there are Drosophila lines available with germline expression of φC31 integrase which allow highly efficient transgene insertion.
In the pUASTattB 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 pUAST 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|
|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|
Our pUASTattB Drosophila gene expression vector is designed to achieve efficient 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 based vector systems such as pUAST.
Potential 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 generation of transgenic Drosophila using the pUASTattB vector system requires the use of specialized host lines carrying attP “landing sites” in their 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: 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.
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.
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.