pUAST Drosophila Gene Expression Vector (User-defined promoter)
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 can be used for achieving ubiquitous, tissue-specific or inducible 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 the helper plasmid encoding the P transposase 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.
The user-defined promoter version of the pUAST Drosophila gene expression vector allows users to select a promoter of their choice from our Drosophila promoter database for driving the expression of their gene of interest (GOI) depending upon their experimental goal. Our Drosophila promoter database offers the following promoter choices: ubiquitous promoters including actin 5C, polyubiquitin and alpha-1 tubulin for driving ubiquitous expression of the GOI; tissue-specific promoters such as Rh2 for driving GOI expression, specifically in Drosophila ocelli and inducible promoters such as Mtn for achieving inducible expression of the GOI with the presence of Cu+.
For further information about this vector system, please refer to the papers below.
|Methods Mol Biol. 420:61 (2008)||The use of P-element transposons to generate transgenic flies|
|Mol Cell Biol. 10:6172 (1990)||Characterization of the actin 5C promoter|
|Mol Cell Biol. 8:4727 (1988)||Characterization of the Drosophila polyubiquitin promoter|
|Nucleic Acids Res. 19:5037 (1991)||Comparison of the alpha 1-tubulin promoter with other Drosophila promoters|
|Genetics. 120:173 (1988)||Analysis of the Rh2 promoter|
|Genetics. 112:493 (1986)||Characterization of the Mtn promoter|
Our pUAST Drosophila gene expression vectors are designed to achieve efficient P transposase-mediated genomic insertion of a GOI. Our vectors are optimized for high copy number replication in E. coli and high efficiency transgenesis of Drosophila lines. The user-defined promoter version of this vector allows users to select a ubiquitous, tissue-specific or inducible promoter for driving the expression of their GOI depending upon their experimental goal.
Flexibility: The user-defined promoter version of the pUAST vector allows users to select a ubiquitous, tissue-specific or inducible promoter for driving their GOI depending upon their experimental goal.
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.
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.
Promoter: The user-selected promoter driving the downstream gene of interest is placed here.
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.