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 Gu+.
For further information about this vector system, please refer to the papers below.
