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Which attP stock should I use?

The advantages of attB/P site specific integration

The original approach to introducing a transgene into the Drosophila genome was to subclone the gene between P-element transposon ends and then inject this construct, plus a helper plasmid expressing P-element transposase, into Drosophila embryos. The P-element transposase enzyme is able to excise the P-element ends, along with any genes between them, from the injected construct and to integrate this DNA into the Drosophila genome.

A major shortcoming of the P-element based approach is that the genomic insertion site of the transgene is unpredictable. This is a problem as the effects on transgene activity of a specific genomic region are unknowable without extensive experimental characterisation. Transgene expression can be influenced by local chromatin conformation or by the presence of specific enhancers sequences. In some cases, insertion close to heterochromatin may lead to variegation in which trangene expression can vary unpredictably from cell to cell.

In attB/P site specific integration, a construct carrying an attB site is injected into a fly stock carrying an attP site at a specific location in the genome. The activity of phiC31 integrase (encoded by a transgene in the injected stock or by a co-injected helper plasmid) recombines the attB and attP sites and integrates the entire construct into the genome. This means that a transgene can be targeted to an attP site at a specific genomic location that has already been tested for any enhancer or chromatin effects.

Which attP stock should I chose for my microinjection?

Ideally, the expression of a transgene inserted at an attP site should not be influenced by local enhancers or chromatin effects. Conveniently, attP sites have been identified on the major Drosophila chromosomes that satisfy these criteria (1). In practice, the landing sites attP18 (X chromosome), attP40 (2nd chromosome) and attP2 (3rd chromosome) might justifiably be considered the default choices for transgene insertion on these chromosomes.

However, other considerations might be important when choosing an attP stock. For example:

Chromosomal location: If, for example, you plan to recombine your transgene onto an FRT chromosome, you will need to select an attP site that is on the same chromosome arm as the FRT site, but sufficiently distant from the FRT to allow recombination to occur at a significant rate. Similarly, recombining your transgene onto a mutant chromosome requires consideration of the relative locations of the attP site and your mutant gene to ensure there is a reasonable frequency of recomination between them.

Previous experiments: Clearly, if you wish to compare the activity of different transgenes it is best that they have been integrated at the same location (attP site) in the genome. Therefore, if previous experiments have used a specific attP site, it may be best to select the same site for future transgenes.

(1) Pfeiffer BD, Ngo TT, Hibbard KL, Murphy C, Jenett A, Truman JW, Rubin GM. Refinement of tools for targeted gene expression in Drosophila. Genetics. 2010 Oct;186(2):735-55

Removal of the integrase transgene

Most of our attP injection stocks carry a PhiC31 integrase transgene which is under the control of a germline promoter (e.g. vas- or nos-). It is good practice to remove the PhiC31 integrase transgene when generating stocks of your insertion. This is not because of the risk of excising your transgene, as the hybrid attB/P sites generated during recombination are no longer a substrate for the integrase. However, removal of the integrase transgene eliminates the possibility of unpredictable effects of integrase expression and the inconvenience of having the integrase transgene floating in your transformant stocks.

In most cases, the integrase transgene in our injection stocks is carried on the X chromosome. Therefore, we initially focus on screening the progeny of injected (GO) males for transformant male (G1) flies. These G1 males, and the transformant stocks generated from them, will no longer carry the integrase X chromosome. If we do not recover this class of transformants, we look for transformants among female progeny of G0 males or among the progeny of G0 females. In this event, removal of the integrase X chromosome will take an additional generation.

 

Contact Information

Tiny fly

E-mail contacts

Simon Collier (Fly Facility Manager): s.collier@gen.cam.ac.uk

General Fly Facility enquiries: flyadmin@gen.cam.ac.uk

Microinjection enquiries: flyinject@gen.cam.ac.uk

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University of Cambridge, Downing Street,
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Phone: +44 (0)1223 765124
Fax: +44 (0)1223 333992

 

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