Responsibilities of the Investigator

• Provide the facility with one or two males (2-4 months old) for each line.
• Important: Each male must be of proven fertility prior to sperm freezing.
• If you require that sperm from more than one male be frozen individually (rather than pooled), submit a requisition for each male as a separate line.
• Although we request a 2-4 month old male, we can process any strain or age of mouse; however, it should be noted that age and mouse strain will affect the fertility of the recovered sperm.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• Sacrifice male(s), harvest epididymides and collect sperm in cryoprotectant medium.
• Sperm will be pooled from all males submitted for a line.
• Freeze and store 10 straws in liquid nitrogen.

Responsibilities of the Investigator

• Have a minimum of five proven stud males available for each line.
• If providing female mice, donors must be between three and four weeks of age, provided in groups of at least five.
• Submit a Request Form in iLabs to initiate your project.

Services provided by the facility

Embryo cryopreservation will be accomplished by mating donor female mice to stud males of the line of interest. Alternatively, in vitro fertilization can be conducted with sperm from one male and oocytes from wildtype females for the production of embryos.

• The facility will superovulate donor female mice and mate to investigator stud males.
• Embryos will be harvested at d 2.5.
• The facility will freeze morula/blastocyst stage embryos in straws with a controlled rate freezer.
• Store straws in liquid nitrogen.
• Thaw test straw to confirm viability of embryos.

Responsibilities of the Investigator

• Provide one male or 2 vials/straws of frozen sperm.
• If providing a male for collection of fresh sperm, the male must be of proven fertility.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• The facility will superovulate wildtype females of the requested strain.
• Harvest oocytes and co-culture with sperm in vitro.
• Transfer resulting two-cell embryos into pseudopregnant recipient females.
• House recipient females and pups to 10 days before transfer to Investigator.

The Transgenic Facility can perform transfer of embryos into pseudopregnant recipient female mice for the purpose of “cleaning” a pathogen-positive colony or to promote colony expansion. 

Responsibilities of the Investigator

• Provide a cohort of five to 10 donor female mice (age TBD by TGIF staff).
• Accompany TGIF staff to your mouse room on superovulation days.
• Have five to 10 stud males available for overnight mating.  Proven breeders are best.
• (Alternatively, provide one male mouse for in vitro fertilization.)

Responsibilities of the Transgenic Facility

• Superovulate investigator donor female mice and pair with males.
• Collect embryos and transfer to recipient females. 
• (Alternatively, order wildtype donor females for superovulation and conduct IVF.) 
• Test pups to ensure that they are specific pathogen free.
• Request transfer to the Investigator’s desired animal room.

The Transgenic Facility provides an initial consultation to all investigators wishing to create a gene-modified mouse or cell line. After the consultation, reagents can be designed and validated. CRISPR reagent validation includes reagent design, reagent validation in embryos or a relevant cell line, and optimization of the PCR reaction for detecting mutations at the target site. For more information, refer to the Fee Schedule or send us an email at tgif@kumc.edu.

In vivo Gene-Edited Mouse Production by CRISPR/Cas9

Responsibilities of the Investigator

• Obtain IACUC approval for your project before requesting service.
• Discuss your intended design and target sequence with the Facility Directors before initiation of the project.
• If your project design involves homology-directed repair, please inform the Facility.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• The Facility will design and order CRISPR reagents (crRNA from IDT) and validate activity in embryos.
• After an active guide or guides have been identified, the Facility will design a targeting construct for knock in projects.
• The Facility will microinject or electroporate RNP with or without a repair template for the production of gene-modified mice.

• The Facility will guarantee the generation of two founders for gene-deletion experiments only. For HDR-based experiments, the Facility will guarantee injection and transfer of at least 250 embryos and/or 30 pups born, although reasonable efforts will be made to generate the mice requested.
• The Facility will genotype all pups resulting from CRISPR-Cas9 injection or electroporation to identify those animals possessing the mutation of interest.
• NOTE: The Facility cannot guarantee germline transmission.

Responsibilities of the Investigator

• Obtain IACUC approval for your project before requesting service.
• Discuss your transgene design with the facility directors before initiation of the project.  Important: when designing your construct, ensure the transgene sequence is flanked by unique restriction enzyme sites to allow for easy removal of the vector backbone.  Isolation of your insert is essential, as inclusion of prokaryotic sequence may interfere with expression of your transgene.
• Provide 25ug of circular plasmid DNA containing your construct at a concentration of 1mg/ml in TE buffer.
• Provide a restriction map and sequence of the construct including size, vector, promoter, gene of interest and/or reporter gene.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• The Transgenic Facility will linearize, purify and quantitate your construct for injection.
• Genotyping will be provided by the facility to identify transgenic founders.
• The facility will guarantee two founders. 
• We cannot guarantee expression and/or transmission of your transgene.

Responsibilities of the Investigator

• Obtain IACUC approval for your project before requesting service.
• Discuss cell source with facility directors before beginning project. For requests to inject ES cells not provided by the Facility (including C57BL/6 cells from KOMP and EUCOMM), please contact Melissa Larson prior to obtaining those cells.  
• Karyotype your cells prior to injection. 
• Prepare cells the morning of injection and submit a single cell suspension on ice, or have the facility culture cells for you.
• Testing for germline transmission is the responsibility of the investigator.
• Submit a Request Form in iLabs to initiate your project.

Services Provided by the Facility

• When requested, the facility will expand and prepare cells for injection.
• Previously selected targeted ES clones will be microinjected into C57Bl/6 blastocysts, after which they will be implanted into pseudopregnant females.
• We guarantee to inject and implant at least 60 blastocysts.
• We cannot guarantee the production of chimeric mice, nor their germline potential.

The Transgenic Facility offers the service of stock development to breed your newly-generated gene-modified founder mice to the F1 generation.  The Facility will mate founders of your choosing to wildtype mice, genotype the resulting pups and confirm by Sanger sequencing.  This service ensures that the investigator receives mice with the transmitted de novo allele.

TGIF staff are available to provide management services for your mouse colonies, including breeding, weaning, culling and collecting biopsies.  Staff can send biopsies to Transnetyx, provide the biopsies to the investigator lab, or genotype in the Transgenic Facility.  All decisions regarding breeding arrangements, weaning and culling are determined by the Investigator; the Transgenic Facility provides the labor.

The Embryo Manipulation Core of the Transgenic and Gene Targeting Facility can accommodate special requests for service, including:

• Embryo collection
• Embryo injection
• Sub-zonal lentivirus injection
• Embryo bisection
• Embryo transfer (mice and rats)
• Vasectomy
• Intrabursal injection
• Artificial insemination

Talk to us about your project!  We will do our best to meet your embryo needs.  Fees will be determined on a per case basis and discussed in advance. Email: TGIF@kumc.edu with you project questions.