Welcome to Fly Facility !
Using our services you can rapidly generate both P-element and site-specific transgenic Drosophila lines carrying different reporter gene constructs, expression constructs and test gene function by dsRNA microinjection. More than 1000 transgenic lines generated and more than 200 dsRNAs injected and analysed. Our device : Quality, Rapidity and Competitive pricing.
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Protocols
Transgenic line production PDF Print

P-element transgenic line production

  • Injections are performed within 2 to 7 days upon arrival of the samples.
  • Survival larvae are picked up in 48 h after injection.
  • Injected larvae are reared in our lab at 25 °C to adulthood in 14 days.
  • 100 crosses is done after injection.
  • Screeni ng and collection of the transformants are done 15 days after crossing.
  • The minimu m turnaround time from reception of DNA samples to delivery of 
      the larvae is 10 days and 6 weeks to get the transgenic flies.

PhiC31 site-specific transgenic line production

PhiC31 integrase transgenesis is based on the site-specific bacteriophage PhiC31 integrase which mediates construct integration between a bacterial attachment site (attB) and a phage attachment site (attP). Because the two sites recognized by the PhiC31 integrase differ and the recombination process mixes the two sites into two different sites (attR and attL),

PhiC31- based integration is irreversible. In practice, plasmids containing attB site and white marker  are injected into strains containing attP-landing site with PhiC31 activity (visit the FlyC31 website for more info) resulting in stable w+ transformants containing your construct-of-interest between attL and attR sites.
 
As an additional advantage the site-specific system allows integration of large DNA fragments using P[acman] system (visit the Bellen lab for more info). 
This system makes use of the combined tools of P1 and BAC construct, recombineering, and PhiC31-mediated trangenesis.
We propose injection of P[acman] vectors into attP landing strains carrying endogenous PhiC31 source developed by the Basler group (see currently available attP lines).
If requested, we can also inject into other attP lines (eg. from Bellen collection) or other lines provided by the customer.
We will put more attP lines available once their afficiency and enhancer trap effect tested.
 
DNA for microinjection PDF Print

DNA Requirements for microinjection

The most critical factors affecting integration efficiency is purity of the DNA.
It is essential that microinjection DNA be absolutely free of contaminants that are toxic to embryos as trace of phenol, ethanol and enzymes.
The efficiency of integration is also influenced by endotoxins plasmids contamination.

Plasmid DNA preparation

Cesium Chloride gradient centrifuge preparation yields the purest plasmid DNA.
We are routinely recommended to use Qiagen Endo Free Plasmid kit (sit: cat.12362),
NucleoSpin TM Extract kit (sit: K3051-1) or NucleoBond AX 500Tip (sit: cat 4003-1).

Preparation of microinjection samples DNA.

DNA concentration must be measured exactly in order that final concentration for injection 4,5 mg/ml can be determined.
Generally we provide the helper plasmid.
We recommend also to use high quality sterile Water for Embryo Transfer, (Sigma W1503), to dilute DNA.
It is also very important that DNA solution do not contain any small particles, which can clog the microinjection needles.

DNA shipping

Send 45µg of plasmids under 2.5 vol of 100% EtOH and 1/20 vol of 3M NaAcet or as precipitated pellet or dissolved in ultra pure water.
In any case indicate very clear your choice.