Partial Services

Partial Services by ingenious

Offering Vector Creation 
& Gene Targeted ES Cell Lines

 

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Partial Services: Vector Creation

Take advantage of our expertise and the services offered by your core facility. Together we design a custom DNA targeting vector for your specific needs.  Our scientists handle the complex cloning and verification steps, then you work with your core facility to produce your custom mouse model.

Donor/Repair Vectors for CRISPR/Cas9 Gene Editing

CRISPR/Cas9 technology has brought rapid progress in mammalian genome editing by increasing the ease and frequency of targeted events. But a limitation is the size of single-stranded oligos that can be readily produced, making genetic modifications spanning larger distances challenging. This rules out efficient production of many popular model types such as conditional alleles and gene knockins. Larger vectors that introduce such modifications require larger homology arms (3kb or more) for efficient recombination, and creating such a vector can be challenging and time-consuming.

Let our experts create the donor vector that you need for your CRISPR project in record time and without the cloning headaches.
Some examples:
  • Any size cDNA insertion vectors
  • loxP (conditional KO) vectors with recombinase sites spaced beyond 1 Kb apart
  • Reporter gene knockin vectors
  • Combinations of any of the above
  • Or contact us to discuss your insertion need

A well-designed targeting vector can serve as a donor/repair vector for use with CRISPR/Cas9, with the backup option of using it in ES cells if direct injection into embryos is unsuccessful.


Gene Targeted ES Cell Lines

ingenious delivers custom gene targeted ES cells. We design and build the targeting vector, electroporate it into our robust ES cells, and subsequently screen the ES cell clones by PCR and Southern Blotting. The targeted clones can be microinjected into blastocysts at your core facility to generate chimeras and subsequently F1 mice.

We guarantee the generation of positively targeted ES cell clones if all work is performed at our facility. This includes all troubleshooting, if necessary, at no additional cost.

Utilizing our FLP ES cell lines alleviates the process for deleting the Neomycin selection cassette, saving you at least 3 months of time, and valuable funding.


STARTING WITH A TARGETING CONSTRUCT PROVIDED BY A THIRD PARTY

We have worked with numerous targeting constructs provided to us by clients who have either generated their own constructs or purchased them from a third party. We can electroporate such constructs into our robust ES cells and provide them to you for screening, or we can perform the screening at our company. If we identify positive clones via PCR and Southern Blotting analysis, we will guarantee germline transmission at our facility, injecting as many times as necessary to produce chimeras and germline confirmed F1 mice.

STARTING WITH KOMP/EUCOMM TARGETING CONSTRUCTS OR TARGETED ES CELL CLONES

The International Knockout Mouse Consortium (includes KOMP and EUCOMM) has created an important resource of targeting constructs, targeted ES cells, and recently, targeted mice. If the targeted mice have not been created yet for your gene of interest, we can use the targeting vector or the targeted ES cell clones from the consortium to generate the mice.

WORKING WITH A TARGETING VECTOR FROM THE CONSORTIUM

We have generated several targeted ES cell lines and targeted mice from KOMP/EUCOMM targeting vectors. We electroporate your chosen construct into our ES cells and screen the resulting clones using PCR and Southern Blotting. If no positive clones are identified, you have the option of terminating the project without incurring any cost. This essentially provides our clients with a “free shot” at obtaining targeted ES cell clones. Once positive clones are identified and confirmed via Southern Blotting, we guarantee germline transmission mice as long as positive clones are injected at our facility.

Protect your investment with ingenious’ guarantee of germline transmission. Search now for your KOMP/EUCOMM targeting vector.

WORKING WITH TARGETED ES CELL CLONES FROM THE CONSORTIUM

We suggest purchasing 3-4 individual clones if possible and performing essential quality control steps to ensure the clones are properly targeted and have high percentage euploidy. We can provide karyotyping and screening services, or those steps can alternatively be completed by the client. Confirmed, high quality clones should be selected for blastocyst injections to generate chimeras, and subsequently the germline confirmed F1 mice.

AFTER YOU RECEIVE YOUR MICE

ingenious provides mouse model service options to address any and all of your needs, but our services do not stop there. Once you receive your model, your next step will be to generate a cohort of mice to utilize for experiments. The generation of experimental cohorts can be challenging, requiring multiple matings, strategic genotyping, and calculated colony scale up. ingenious offers prediction tools and expertise for developing the cohorts you need, as well as cryopreservation services for protecting your investment. Read more about our post project support services.

Further Reading

https://www.researchgate.net/publication/283511253_Homology_Arms_of_Targeting_Vectors_for_Gene_Insertions_and_CrisprCas9_Technology_Size_Does_Not_Matter_Quality_Control_of_Targeted_Clones_Does

https://blog.benchling.com/how-to-design-homologous-recombination-template-for-crispr/



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