Our pUASTB vector system is highly effective for generating transgenic flies and controlling transgene expression. This system has the capability to utilize either the Drosophila P-element transposon system (like pUAST) or the bacteriophage φC31 integration system (like pUASTattB), and can be used for achieving ubiquitous, tissue-specific or inducible transgene expression. Thus, the pUASTB vector system can be used for either P transposon-mediated or φC31 integrase-mediated insertion of your gene of interest. In either case, the user selected gene of interest is cloned into pUASTB in a region bracketed by two P-element terminal repeats and near an attB recombination site.
To utilize P transposon-mediated insertion, the pUASTB plasmid and a P transposase-expressing helper plasmid are co-introduced into host cells or embryos. As a result, the transposase produced from the helper plasmid recognizes the two P-element terminal repeats on the pUASTB plasmid, and inserts the flanked region including the two P-element terminal repeats into the host genome. P transposase-mediated insertion occurs without any significant bias with respect to insertion site sequence.
To utilize φC31 integrase-mediated insertion, the pUASTB plasmid and a φC31 integrase-expressing helper plasmid are co-introduced into host cells or embryos containing attP landing sites. The φC31 integrase mediates irreversible recombination between attB and attP sites, resulting in the linearization and integration of the pUASTB vector into the host genome.
The mini white gene on the pUASTB 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.
The user-defined promoter version of the pUASTB 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.
|Methods Mol Biol. 420:61 (2008)||The use of P-element transposons to generate transgenic flies|
|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|
|Genetics. 166:1775 (2004)||Use of pUASTB to construct transgenic Drosophila|
|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 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 pUASTB Drosophila gene expression vectors are designed to achieve either P transposase-mediated or φC31 integrase-mediated genomic insertion of a gene of interest. 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 their GOI depending upon their experimental goal.
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.
Flexibility: The user-defined promoter version of the pUASTB vector allows users to add a ubiquitous, tissue-specific or inducible promoter for driving their GOI depending upon their experimental goal.
High efficiency: Achieving germ-line transgenesis using φC31 integrase vectors is more efficient than P-element based vector systems such as pUAST.
Random genomic insertion: If P transposase-mediated insertion of pUASTB is used, random integration of P-elements can make it difficult to map genomic insertion sites, and genomic position can affect transgene expression. Additionally, transgene insertion into genes or regulatory elements within the genome can affect endogenous genes.
Technical complexity: The generation of transgenic Drosophila requires embryonic injection and fly husbandry, which can be technically difficult.
Requires attP insertion sites: The use of φC31 integrase-mediated insertion of pUASTB requires the use of specialized host lines carrying attP “landing sites” in their genome.
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: Drosophila 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. φC31 integrase can catalyze site-specific integration of attB-containing plasmids into attP-containing docking or landing sites that have been introduced into host genomes.
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.