The transgenes insert at random positions in the genome occurs. The plasmid must contain the inverted repeats of the P element as well as the mini-white gene to identify the positives.

We require 10 ug of DNA  in a volumen of 10ul for each construct injected. It is not necessary for the user to provide the DNA helper.

Strains available in the service:

– w, yw

Transgene insert at well-defined receptor sites in the genome,  containing attP sequences.

Transgenes should be cloned into vectors containing attB sites and the mini-white gene to identify the positives.

DNA preparations should be of high quality and at a minimum concentration of 300 ng/ul in a minimum volume of 20 ul.

Strains available from the service:

–2L y[1] M{vas-int.Dm}ZH-2A w[*]; M{3xP3-RFP.attP’}ZH-22A B#24481

–2R y[1] M{vas-int.Dm}ZH-2A w[*]; M{3xP3-RFP.attP}ZH-51D B#24483

–3L y[1] M{vas-int.Dm}ZH-2A w[*]; M{3xP3-RFP.attP’}ZH-68E B#24485

–3R y[1] M{vas-int.Dm}ZH-2A w[*]; M{3xP3-RFP.attP}ZH-86Fb B#24749

We can inject mixtures of nucleic acids containing gRNAs, plasmids encoding gRNAs and ssDNA or plasmid templates for CRISPR/Cas9-assisted genomic editing. The strain we used expresses Cas9 in the germline under the control of the nanos promoter.

The service cannot provide technical support regarding the experimental design or the different possible technical approaches.

The user must provide us with the nucleic acid mixture ready for injection. The service recommends: 3ug guideline RNA and 10ug donnor in 20ul volume.

Strains available from the service:

-25C y[1] v[1] P{y[+t7.7]=nos-phiC31\int.NLS}X;P{y[+t7.7]=CaryP}attP40 B#25709

-68A y[1] sc[1] v[1] P{y[+t7.7]=nos-phiC31\int.NLS}X; P{y[+t7.7]=CaryP}attP2 B#25710

–Noscas9: y[1] M{w[+mC]=nos-Cas9.P}ZH-2A w[*]  B#54591

–Vascas9 RFP-:y[1]M{GFP[E.3xP3]=vas-Cas9.RFP-}ZH-2Aw[1118] B#55821

 When users need to inject samples into specific strains from their laboratories, our facility will charge for the husbandry of these strains

DNA PREPARATION

We recommend DNA purification using the Qiagen kit or similar. The concentration and purity of the DNA should be analyzed prior to shipment.

SCHEDULE

For P-element-mediated or PhiC31 integrase-mediated transgene generation, we inject 300 to 400 embryos per plasmid. In the case of the CRISPR/Cas9 method, we inject 500 to 600 embryos per plasmid.

The surviving larvae (75 to 150) are sent to the user 3-4 days after injection.

Users will be responsible to screen for transgenic lines in their laboratories. If no transformant is obtained, we will repeat the injection once, with priority and at no additional cost.

SHIPMENTS

Shipments of larvae after injection are at the users’ expense. Multiple carriers are available.

SAMPLE SHIPPING INSTRUCTIONS

Samples should be sent to the following address:

Mar Casado/Nuria Esteban
Centro de Biología Molecular Severo Ochoa
C/Nicolás Cabrera 1
28049, Madrid
Teléfono: +34-911964610

The facility requires users to fill in the following forms. If this is the first time you use our service, you must also sign the legal agreement.

DOCUMENTS

• Agreement
• General condictions
• Order form
• Invoice information