gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
pUAST Drosophila Gene Expression Vector
Our pUAST vector system is a well-characterized and highly effective system for generating transgenic flies and controlling transgene expression. This system is derived from the commonly used Drosophila P-element transposon and incorporates a strong Gal4-inducible promoter to regulate transgene expression.
The complete pUAST system consists of two vectors, both engineered as E. coli plasmids. One vector referred to as the pUAST plasmid, contains two P-element terminal repeats bracketing the region/gene to be transposed. The other vector, referred to as the helper plasmid or transposase plasmid, encodes the P transposase.
When the pUAST and the transposase plasmid are co-injected into target cells, the transposase produced from the helper plasmid recognizes the two P-element terminal repeats on the pUAST plasmid, and inserts the flanked region including the two P-element terminal repeats into the host genome. Insertion occurs without any significant bias with respect to insertion site sequence.
The P-element is a class-II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class-I transposons move in a copy-and-paste manner.) The transposition creates 8 bp direct repeats at the integration site in the genome.
The pUAST system is commonly used to generate transgenic flies by co-injecting the pUAST and helper plasmids into Drosophila early embryos. P transposase-mediated recombination between the two P-element terminal repeats leads to germline recombination events which produce transgenic offspring carrying the user’s gene of interest. The P transposase will only be expressed for a short time, and with loss of the helper plasmid, the integration of the transposon in the host genome becomes permanent. The mini white gene on the pUAST vector encodes for eye color and acts as a marker for the identification of transgenic flies which have undergone successful recombination of the transgene. PCR or other molecular methods can also be used to identify transgenic cells or animals.
In the pUAST 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|
|Methods Mol Biol. 420:61 (2008)||The use of P-element transposons to generate transgenic flies|
Our pUAST Drosophila gene expression vectors are designed to achieve efficient P transposase-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.
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.
Random genomic insertion: The random integration of P-elements can make it difficult to map insertion sites, and genomic position can affect transgene expression. Additionally, transgene insertion into genes or regulatory elements within the genome can affect endogenous genes.
Moderate efficiency: Achieving germ-line transgenesis using P-element vectors is generally less efficient than φC31 integrase-mediated systems such as pUASTattB.
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.
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: 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.
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.