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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Services
Transgenic lines PDF Print

We are proposing the following services:

Purification of DNA samples for injection - 40 €   
P-element Basic - 180 €
P-element Extended - 230 €
Site-specific Basic - 180 €
Site-specific Extended - 230 €
 

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Purification of DNA samples for injection - 40 €:

As the quality of DNA sample has an important influence on efficiency of transgenesis we propose plasmid purification to standardize this step. We are using Qiagen Endo Free Plasmid purification kit ensuring that DNA is free of endotoxins and other contaminants such as trace of phenol, ethanol or enzymes.


P-element Basic - 180 €:

Accurately purified DNA sample of the P-element construct for injection is provided either by customer (see Protocols ) or is purified by our team as a service option (see above).

Prior to injection, we check DNA concentration, ethanol precipitate and mix with the helper plasmid (we provide).
Basic service includes injection of the P-element construct into 250 w1118 embryos and sending back the survival larvae in vials.
You are responsible for identifying your own transformants and balancing them.
We guarentee at least 3 transformant lines for constructs of reasonable size (less than 13 kb total plasmid size).

We developed a user-friendlyImage Exemple Tracking-System that allows you to follow the progress of transgenesis with your sample.

P-element Extended - 230 €:

We will rear the injected larvae to adulthood following the Basic services. Cross the G0 injected flies to w1118 virgins or males (usually 100 individual crosses). Identify and collect the transformants. The collected transformant flies (at least 3 independent lines) will be sent to the customer and original vials will be maintained in our lab for about two weeks. If your DNA for injection is larger than 13 kb we can get less than 3 independent transfotmants because large constructs are more difficult  to insert into the genome.

As for basic service you can follow the progress of transgenesis using our on-line Image Exemple Tracking-System .

Site-specific Basic - 180 €:

Purified DNA sample of an attB containing vector is provided by customer (actually we do not propose purification service for site-specific vectors). Depending on the attP strain used 200-300 embryos are injected. For some P[acman] system injections, we will use embryos from the crosses between the selected docking-site strain and the PhiC31 integrase-source strain. For injections into strains from FlyC31 collection (Basler lab) containing the PhiC31 source no cross is required. In any case we will collect the survival embryos and send, the G0 larvae embryos. You are responsible for identifying your own transformants. We do not guarantee transformants in Basic service however we can suggest « landing » strains that in our hands are relatively efficient.

As in other services you can use Image Exemple Tracking-System to follow the progress of transgenesis.

Site-specific Extended - 230 €:

This service in addition to Basic service includes identification of site-specific transformants. We will inject your attB DNA sample into 200-300 embryos, back-cross survival adults to yw and ship the transformants to you. We guarantee at least 1 independent transformant line but send all found in the screening process.
"1 independent G1 transformant ª due to the fact that all transformants have the same insertion site and you need only one line for analysis.

You can follow the progress in generation of transformants by logging to the Image Exemple Tracking-System .   

PhiC31 integrase-mediated site-specific transgenesis

We can order the stocks containing the docking sites that are available from the Bloomington Drosophila Stock Center. (Supplementary time)
To generate transgenic animals we can use flies with endogenous PhiC31 source or co-injecting the PhiC31 mRNA. It is costumer’s choice.
In our hands the best transformation score we have with Bloomington lines

No. 24749, 24482, 24872 and 24871(genotypes below).

y1
M{vas-int.Dm}ZH-2A w*; M{3xP3-RFP.attP}ZH-86Fb
y1 M{vas-int.Dm}ZH-2A w*; M{3xP3-RFP.attP'}ZH-51C.
y[1] M{vas-int.Dm}ZH-2A w[*]; PBac{y[+]-attP-3B}VK00037
y1 M{vas-int.Dm}ZH-2A w*; PBac{y+-attP-3B}VK00033

 
dsRNA injection PDF Print

How it works

RNA interference offers a possibility for rapid analysis of gene function. We developed and standarized direct dsRNA injection method into embryos carrying GFP transgenes expressed in a tissue-specific manner.

We can provide GFP lines expressed in the embryonic muscle (MHC-GFP) in the heart (Tin-GFP, Him-GFP) and in the CNS (Elav-GFP) so that the analyses of phenotypes in these tissues can be performed. Other tissue-specific GFP lines provided by customers can also be used.

To determine effects of attenuation of gene function we inject 100 embryos at blastoderm stage (ventrally, when mesoderm and CNS are targeted). dsRNA of 400-500 nt is synthesized on PCR-generated T7-flanked DNA templates using T7 RNA Pol (see protocols). To avoid off target effects, two distinct templates are generated and two distinct dsRNAs are injected for a single gene.

As a result of injection we determine the percentage of lethality and we document the changes in GFP pattern in embryos with phenotypes (approximately 10 photos per gene and/or time lapse analysis if requested). CELL® - Optigrid and confocal scanning microscopy is used for documentation.

Usually dsRNA injection and documentation of phenotypes for one gene takes 1 week. Design, synthesis of primers and production of dsRNA probes take additional 2 weeks.

For RNAi screen purposes (large number of genes to be tested) contact our platform staff .

There are two levels of dsRNA services we propose:

Basic

Customer provides dsRNA probes, which are injected and receives documentation of phenotypes.
Non-Profit academic users - 200€ / gene

Extended

Customer indicates the gene of interest and we are in charge to design, produce and inject dsRNA and document the phenotypes.
Non-Profit academic users - 300/ gene

 

MHC-GFP - muscle-specific GFP line used for testing muscle gene function, time lapse experiment showing muscle contraction