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Time Table and Pricing - Gene Targeting Core

Time Table
We plan to do three targeting runs per year, Jan, May and Oct, producing four mouse strains per run. For example, at the moment (October 8, 2015) we are generating TALENs for four different  genes. We know that null alleles are viable on a WT background, but wish to introduce null alleles on strains with multiple genetic modifications. This approach will be faster than conventional breeding strategies.

In this case, will generate mutations on a C57BL6/J mouse strain with a hypomorphic Pkd1 allele, Pkd1R3277C/R3277C (PMID:23064367). As fibrocystin is also thought to be a member of the polycystin protein complex, these mice will also have an epitope tagged WT form of Pkhd1, Pkhd1pk(+)/pk(+). The Pkhd1pk(+) allele has two IPNPLLGL tags on the extreme N-terminus of fibrocystin allowing the investigator to follow fibrocystin in the urine or tissues using the V5-Pk1 monoclonal antibody (Serotec) (PMID:22021705). Pkd1R3277C/R3277C can be regarded as a sensitized Pkd1 strain, slowly developing renal cysts over a 9 month period. Mutations in genes interacting with the polycystin complex will exacerbate the cystic phenotype. In cases where the mutation ameliorates the cystic phenotype the Pkd1R3277C/R3277C mice can be crossed onto Pkd1 null heterozygotes to generate Pkd1R3277C/- mice, that have severe renal disease.

We will commence another mutagenesis run early in Jan 2016 focusing on WT C57BL6/J mice and we have four slots open for this targeting. Later targetings will be applied to WT or PKD sensitized strains, depending on demand.
 
Pricing
While the core will be subsidized by the NIH P30 grant, this will not be enough to cover all costs and thus we plan to ask for some remuneration. The actual cost of a microinjection at KUMC is $4,000 and so we will subsidize internal users $1,000 per injection; external users will get injections at cost. We have permission to use the CRISPR-Cas9/TALEN system under a Uniform Biological Material Transfer Agreement (UBMTA) from Addgene providing that we do not make a profit and the TALENs and animals are used for academic purposes only.

Mutations will be detected using high resolution melt analysis (HRMA) on pup tail DNA. Mice positive for a change will be sequenced. We will attempt to supply two different alleles per targeting.

  Service Internal Users External Academic Users
Production of TALEN pair

$50 per pair

$100 per pair

Production of RNA for injection

$50 per pair

$100 per pair

RNA microinjection in WT zygotes

$3,000 200 zygotes

$4,000 200 zygotes

RNA microinjection in custom strain

$4,000 200 zygotes

To be negotiated

HRMA on pups

$100 per 10 pups

$150 per 10 pups

Sequencing

$10 per pup

$15 per pup

Storage of embryos

$500 per strain

$600 per strain

Storage of sperm

$500 per strain

$1,000 per strain

 

Contact Information
Dr. Christopher J. Ward MBChB, PhD
Associate Professor of Nephrology
Mail Stop 3018
3901 Rainbow Blvd
Kansas City KS 66160
Tel: 507-226-4642
cward6@kumc.edu



Last modified: Aug 13, 2018
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