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 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.
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.