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Indiana University School of Medicine
Indiana University School of Medicine
Service Cores
Procedures for the genome editing core, providing investigators access to state-of-the-art genome editing technologies.

Procedures

For the generation of genetically modified mouse models and cell lines, fill out the Genome Editing Center's Project Information form and the Model Registration form. Send both back to spellet@iu.edu and destthom@iu.edu. The Genome Editing Center will inform IBC and IACUC of new models being generated.

Upon receipt of the Project Information form, the Genome Editing Center will design a targeting strategy and produce a Statement-Of-Work (SOW). The SOW contains information detailing the targeting strategy, the genotyping strategy, and other important information. A SOW fee will then be charged to the investigator. This fee serves as deposit and will be deducted in full upon completion of the model. This fee will not be reimbursed should the investigator decide not to pursue the project with the center or use a la carte services for the generation of the model.

For traditional transgenic core services, fill out the Transgenic Services form and send the information back to destthom@iu.edu. If you are using our pronuclear injection service, please fill out Model Registration form and send the form to spellet@iu.edu and somiacuc@iu.edu (IUPUI) or biacuc@indiana.edu (Bloomington). Make sure the mouse model engineered is listed on your IBC and IACUC protocols.

Project Information form

Model Registration form

Transgenic Services form

Model generation services

For the generation of mouse models with user-defined mutation, the Genome Editing Center will:

  • Design a targeting strategy.
  • Generate reagents used to create the mouse model.
  • Test genotyping strategy.
  • Generate founder mice.
  • Genotype founder mice.
  • Analyze founders for off-target cleavage (when using CRISPR technology for the generation of the model).
  • Test germline transmission of the genetic modification.
  • Cryopreserve the mouse model (sperm, no additional charges until the completion of the fiscal year).
  • Deliver the mouse model and related documentation to the investigator.

The Genome Editing Center guarantees the generation of the mouse models unless the mutation is not compatible with embryonic development or is lethal early in the life of the animal. The center reserves the right not to take on projects with low chance of success. The center provides C57BL/6 mice. If an investigator needs other strains, he or she will need to pay for them.

For the generation of cell models with user-defined mutation, the Genome Editing Center will:
  • Design a targeting strategy.
  • Generate reagents used to create the cell model.
  • Test genotyping strategy.
  • Transduce cells and isolate clones.
  • Genotype clones.
  • Cryopreserve clones.
  • Deliver the cell model(s) and related documentation to the investigator. The core will deliver all positive clones to investigators along with up to 3 control clones. Control clones are clones that have gone through the engineering process but remained unmutated.

Several factors can affect the engineering process in cell lines. These include:

  • transduction efficiency of the parental cell line,
  • the ability of the cell line to perform homologous recombination,
  • clonability,
  • polyploidy, and
  • the nature of the mutation engineered.

Consequently, if the user wishes to perform genetic engineering in a cell line the Core has never worked with, a series of tests will be performed by the Core prior to engineering the cell line model(s). Optimization fee will apply (see optimization rate under transformed cell line). The Genome Editing Center will test:

  • the clonability of the parental cell line, and
  • transduction efficiency of plasmid DNA and HDR template

Clonability will be tested by serial dilutions. The presence of single cell clones must be visible and growing after 7 and/or 14 days in culture for the Genome Editing Center to engineer the cell line. Transduction efficiency will be tested using 64 electroporation conditions. A 20% transduction efficiency requirement must be met for the Genome Editing Center to engineer the cell line.

If the parental cell line passes these tests, the Genome Editing Center will continue with the engineering process. If the parental cell line does not pass these tests, the Genome Editing Center will communicate with the investigator and determine the best course of action.

During the engineering process, the Core will test:

  • whether the parental cell line is amenable to homologous recombination, and
  • whether the engineered mutation is toxic or prevent cell growth.

Homologous recombination will be assessed by introducing a small DNA segment encoding a restriction enzyme site at a specific locus. The presence of the restriction enzyme site will be assessed by PCR amplification of the locus and digestion. This will be performed 48 hours after transduction on bulk sorted cells. Visible digestion products must be obtained for the Genome Editing Center to engineer the mutation. If no recombination is visible, the Genome Editing Center will contact the investigator and determine the best course of action. If the project is terminated, the core will keep the SOW fee ($2,000) to cover for its expenses (allele design, targeting construct, oligonucleotdies, genotyping, flow cytometry, labor, etc.).

If the investigator decides to pursue the engineering process, 480 clones will be single cell sorted. Proliferating clones (~10%) of sorted clones), will be analyzed at the genomic level. 

Toxicity will be tested by analyzing cells for the presence of the desired mutation immediately after sorting (48h after transduction) and following clonal propagation. If the mutation is present immediately after sorting but not in the clones generated, the Core will consider this mutation as toxic to cells.

The Genome Editing Center does not guarantee the generation of the cell line model if the parental line does not pass these tests, is polyploid (> 2 copies of the gene), or if the mutation is toxic or prevents cell proliferation. Full payment is expected, regardless of the outcome. The Center reserves the right not to take on projects with low chance of success.

Potential off-target editing can be assessed by the Genome Editing Center upon request by the investigator. Up to 5 potential off-target cleavage sites will be analyze. Additional charges apply (see Transformed cell line, Off-target analysis). 

Traditional transgenic services

For pronuclear injection, investigators provide the DNA construct to the Genome Editing Center. The center will:

  • Gel purify traditional transgene constructs.
  • Harvest single cell embryos.
  • Microinject the embryos with the transgene or CRIPSR construct.
  • Transplant the microinjected embryos into pseudo-pregnant females.
  • Perform cesarean sections as required.
  • Provide the resulting pups at weaning age to the investigator for analyses.

The center guarantees the generation of 10 births or one founder (whichever comes first) when using C57BL/6. embryos (historic average is 14 births per round).

The Genome Editing Center reserves the right not to take on projects with low chance of success. The center provides C57BL/6 mice. If an investigator needs other strains, he or she will need to pay for them.

For sperm cryopreservation with verification, investigators provide a minimum of two male mice. The Genome Editing Center will:

  • Harvest sperm from the donor
  • Produce 20 straws of frozen sperm
  • Verify successful freezing via in vitro fertilization using the thawed sperm and donor eggs from the relevant strain of mice; verification entails development of 2-cell embryos following in vitro fertilization

The verification is the guarantee for this service.

For sperm cryopreservation without verification, investigators provide a minimum of two male mice. The Genome Editing Center will:

  • Harvests sperm from the donor
  • Produce 20 straws of frozen sperm

The core guarantees mobile sperm upon test thawing. Cryopreservation activities are based on a good faith agreement between the investigators and the Genome Editing Center. Thus far, the core has not encountered any difficulties in the recovery of cryopreserved samples which it has generated. However, we cannot guarantee that all samples can be recovered following long-term storage.

For in vitro fertilization and embryo transfer, investigators provide either frozen sperm samples or a minimum of two living mice. The Genome Editing Center will:

  • Thaw the frozen sperm or harvests fresh sperm from the donor Performs in vitro fertilization
  • Cultures embryos to 2-cell stage
  • Transplant the 2-cell embryos into pseudo-pregnant females Performs Cesarean sections as required
  • Provide the resulting pups at weaning age to the investigator for analyses.

If no births are obtained, a second round (optimized based on the results from the first round) will be performed for no additional charge. Subsequent efforts will require additional billing.

For embryo thaw and transfer, investigators provide the frozen embryos. The Genome Editing Center will:

  • Transplant the 2-cell embryos into pseudo-pregnant females
  • Perform Cesarean sections as required
  • Provide the resulting pups at weaning age to the investigator for analyses

If no births are obtained, a second round (optimized based on the results from the first round) will be performed for no additional charge. Subsequent efforts will require additional billing.

Cryopreservation activities are based on a good faith agreement between the investigators and the Genome Editing Center. Thus far, the core has not encountered any difficulties in the recovery of cryopreserved samples which it has generated. However, we cannot guarantee that all samples can be recovered following long-term storage.