The advantage of these approach over ES cells targeting is cost, time to generate, and the ability to generate in the strain the investigator desires.

Common examples of this services:

• Gene knockout – injection of CRISPR/Cas9 RNP to target important domains or 5’ exon. Goal is to create a frame shift such that a functional protein will not be generated.
• Generation of a point mutation in a gene - injection of CRISPR/Cas9 RNP plus a targeting oligo such that homologous recombination will generate the desired nucleotide alterations.
• Knock-in of exogenous DNA – one example would be insertion of a GFP or CRE into an exon of a gene to acquire GFP or Cre under the control of that gene’s endogenous promoter. This includes injection of CRISPR/Cas9 RNP plus a targeting vector such that homologous recombination will generate the desired DNA insertion.
• Rosa26 knock-in – this is often used if an investigator wants to overexpress a cDNA of interest, either under the endogenous ubiquitous Rosa26 promoter (low to medium expression), ubiquitous CAGG (high expression), or a spatial or temporal specific promoter the investigator desires. This will have a single copy insertion into the Rosa26 safe harbor genomic locus allows for ubiquitous expression. This includes injection of CRISPR/Cas9 RNP plus a targeting vector such that homologous recombination will generate the desired DNA insertion.
• Conditional allele generation – This essentially involved injection of 2 CRISPR/Cas9 RNP plus a targeting vector such that homologous recombination will generate an allele with a LoxP site on each side of an exon.
• For other genomic editing projects, contact us to discuss design and cost.

Service includes:

• Selection, purchase and testing of gRNA.
• Design and purchase of single-stranded oligos for generation of point mutants.
• Free consultation on targeting vector design, outlining necessary components.
• Generation of single-stranded DNA from investigator-supplied targeting constructs.
• Injection of 150 fertilized oocytes and oviduct transfers.

Note: There is no additional cost if injected into C57Bl/6J or FVB/n mouse strains. Requirements for alternative strains will result in additional costs for colony establishment.

• PCR screening of F0 to identify positive animal(s).
• F1 PCR-positive progeny will be transferred to the investigator.

Service does not include:

• Cloning and generation of targeting vectors. We prefer to assist in targeting vector design and are happy to meet with you to discuss the best design for your project. For labs that do not routinely do molecular biology, we encourage the use of gene synthesis services. For this we will obtain a quote for synthesis of the vector, and the investigator will be responsible for this cost.
• For knock-in based transgenes, we provide F1 positive animals, but it is the investigators job to validate proper insertion and conduct all experiments involving the animal.

This approach has higher success that targeted insertions (for example Rosa26 targeting) however expression is influenced by genomic environment and insertion copy number. This approach often generates single loci alleles with 10-350 concatemer insertions.

• Free consultation on vector design. Note, the vector must have a unique restriction enzyme site outside of the area of interest for linearization of the DNA.
• Purification of DNA for injection from investigator-provided construct.
• Injection of 150 fertilized oocytes and oviduct transfers.
• PCR screening of F0 to identify positive animal(s).
• F1 PCR-positive progeny will be transferred to the investigator.

• Cloning and generation of vectors. We are happy to meet with you to discuss the best design for your project. For labs that do not routinely do molecular biology, we encourage the use of gene synthesis services. For this we will obtain a quote for synthesis of the vector, and the investigator will be responsible for this cost.
• We provide F1 positive animals, but it is the investigators job to validate transgene expression and conduct all experiments involving the animal.

The investigator is responsible for generating the targeting vectors following a consultation on design.

• Vector must have a unique restriction enzyme site outside of the arms of homology (and TK if included) for linearization of the DNA.
• We also suggest one arm being no larger than 1 kb to allow for PCR verification of proper targeting.
• For labs that do not routinely clone, we suggest using gene synthesis services and can assist you in design submission.

We have two tiered services:

• The investigator can PCR screen and identify clones to proceed with for model generation.
• For an additional fee, the Core will PCR screen clones across the short homology arm to identify correctly targeted clones.
• Once the desired clones are identified we will expand up to 2 clones and freeze 5 vials of each for preservation.
• Additional clones can be expanded for an additional fee.

ES cells can be acquired from ES targeting in TGEM, other investigators, or repositories. If from outside UAB, please have them shipped directly to TGEM at:

• TGEM
• 1918 University Blvd.
• MCLM 925
• Birmingham, AL 35233-0005
• Phone: (205) 934-7207

For the Sperm Cryopreservation the lab should provide two males between 2-12 months of age, preferably proven breeders.

The procedure can usually be scheduled for the Tuesday in the week following completion of the submission form.

The male animals should be brought or transferred to MCLM 925 no later than 10am on the day of the scheduled procedure. You can bring them or arrange a transport using ARP using the form found on the ARP website.

Motility of thawed sperm will be tested.

Includes transfer of two-cell embryos to up to six recipient females. In general, we expect ~50 embryos to be transferred but depends on the quality of the sperm.

Standard IVF is with C57Bl/6J or FVB/n mouse strains. Requirements for alternative strains will result in additional costs for colony establishment.

• Friday - 0.1 ml PMS given to each female mouse donor by IP injection between 11-2pm
• Sunday - 0.1 ml HCG given to each female mouse donor by IP injection at the same time PMS was given. Females will then be mated overnight to males of your choosing, preferably 1:1.
• Monday - Before 10am separate matings and check females for copulatory plug.
• Tuesday - Plugged females brought to MCLM 925 before 10 AM for cryopreservation.

Viability of thawed embryos will be tested.

Transfer of fertilized oocytes to up to six recipient females.

Resulting pups will be transferred to requestor upon weaning.

These fees cover the cost of weekly maintenance frozen storage.

We are offering genome editing service (knockout, knock-in of exogenous DNA with selectable marker, or nucleotide alterations) of human IPSC (so far, we have successfully worked with PGP1 IPSC) and human ES (so far, we have successfully worked with H9 cells). We can discuss projects working with other cell lines.

This involves 1) guide design and evaluations; 2) electroporation and single cell clone isolation; 3) PCR based identification of positive clones.

For lines pre-established and maintained in TGEM, we charge a $150 restocking fee per vial