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Embryonic Stem Cells and Mouse modelling (SCMM) at IGB-ABT, CNR

The SCMM at the Institute of Genetics and Biophysics Adriano Buzzati-Traverso runs a service of genome editing into Embryonic Stem Cells (ESC) by means of CRISPR/Cas9-based technology and homologous recombination.

The completion of the work will depend on the waiting list, the service works on a “first come first served” basis.
Fees should be paid in advance once the request is accepted.

 

Construct design and generation


The strategy and the targeting constructs/vectors for the generation of the mutagenesis required (knock-out, conditionals, point mutation, targeted Cre or CreER deleters, gene replacement, etc...) will be agreed with the user.

a) Via cloning into vectors suitable for classical Homologous Recombination:

Homology arms, suitable for PCR screenings, will be cloned into targeting vectors provided by the facility. Generation of the targeting vector will (may) require commercial BAC clones.

This procedure should approximately take 1-2 months.

FEES: (Request Form)

For IGB-ABT users: 3.000,00

For external users:
5.000,00 (+VAT)

b) Via CRISPR/Cas9 Technology:

Targeting molecules will be generated by the facility according to the request (i.e. knock-out, gene replacement, point mutations, etc..)

This procedure should approximately take 1 month.

FEES: (Request Form)

For IGB-ABT users: 1.800,00

For external users:
3.000,00 (+VAT)

 

Generation of ESC clones for blastocyst injection


a) Via Homologous Recombination:

The facility service includes the electroporation and isolation of heterozygous mutant ESC clones for the generation of transgenic mouse models.

The fee includes the screening of 200 ES cell clones, screened by PCR and validated by Southern blot. For Southern blot analysis, the user  has to provide suitable and tested probes, as agreed with the facility.

The homologous recombination procedure should approximately take 2-3 months

FEES: (Request Form)

For IGB-ABT users: 3.000,00

For external users: 6.000,00 (+VAT)

b) Via CRISPR/Cas9 in ESCs:

The facility service includes transfection and isolation of ESC mutant clones.

The fee includes the screening of 100 ES cell clones by PCR-based analysis, which will also be used for further validation of positive clones.

The CRISPR/Cas9 procedure should approximately take 1-2 months

FEES: (Request Form)

For IGB-ABT users: 1.500,00

For external users: 3.000,00 (+VAT)

 

Microinjection


The injection fee is to be considered per number of injected blastocysts (n=80). The fee also covers animal purchase, cell culture costs and maintenance of up-to-six-week old chimeras.

FEES: (Request Form)

For IGB-ABT users:
2.500,00

For external users:5.000,00 (+VAT)

 

Germline trasmission


The facility can provide breeding of chimeras for germline transmission.

The service includes the purchase of wild type mice of the requested genetic background, housing, mating of chimeras, genotyping of the offspring.

Germline transmission is expected within 4 months.

FEES: (Request Form)    
For IGB-ABT users:
1.000,00

For external users: 
4.000,00 (+VAT)

 

Breeding, maintenance and genotyping of mutant mouse colonies


For the transgenic mouse lines generated by the facility, a service of breeding, maintenance and genotyping of mutant colonies is offered for the desired number of mice.

FEES: (Request Form)

For IGB-ABT users:    
0,70/cage/day

For external users:  1,00/cage/day (+VAT)

 

Zygote injection


The facility can provide a service of microinjection of CAS9 mRNA/single-guide RNAs (sgRNAs) into zygotes for the production of transgenic knock-out mouse lines (insertion/deletion, indels) mutations.

FEES: (Request Form)

For IGB-ABT users:     3.000,00

For external users:  
6.000,00 (+VAT)

 

Strain Rederivation


The facility requires three males of proven fertility and genotype, which will be mated in our quarantine space with females of inbred/hybrid/outbred genetic background, according to each customer’s requirements.

The service includes the purchase of embryo donor females, fosters, and maintenance of the offspring until weaning.

Strain rederivation is expected in approximately 2 months.

FEES: (Request Form)

For IGB-ABT internal users:
700,00

For any external user: 1.500,00 (+VAT)

 

Generation of homozygous mutant ESC models


a) Via Homologous Recombination in ES cells:

The homologous recombination procedure to generate homozygous ESCs should approximately take 6-8 months, and includes two rounds of targeting, each followed by removal of the selection cassette.

For Southern blot analysis, the user  has to provide suitable and tested probes, as agreed with the facility.

FEES: (Request Form)

For IGB-ABT users: 6.000,00

For external users:
10.000,00 (+VAT)

b) Via CRISPR/Cas9 in ESCs:

The fee includes the screening of 150 ES cell clones by PCR-based analysis of correct targeting/mutagenesis.

FEES: (Request Form)

For IGB-ABT users:
2.500,00

For external users:
6.000,00 (+VAT)