Genetically Engineered Rodent Models Core Services

Sperm cryopreservation is an efficient, reliable, and cost-effective means of safeguarding and maintaining stocks of critical GEM resources. Investigators prepare 1-3 males between the age of 3 –7 months for each mouse line to be preserved. Sperm is cryopreserved in straws and subsequently test via in vitro fertilization for motility and fertilization capacity.

What will happen

1. The investigator will submit a request form through iLab, filling out detail information on the number of males, ages, and any known reproductive defects. An account number must be provided at this time.
2. The core will review the investigator-provided information and approve the request. The core will schedule the cryopreservation and confirm it with the investigator.
3. The investigator will either deliver the mice to the biosafety hood in to ABBR R119A or have the mice transferred to the core by the CCM transfer system. The investigator needs to write the iLab request number on the cage card and leave the PI’s census card on the cage. If the mice are from BCM satellite locations, the mice must be delivered in a cardboard box (provided by CCM) or transferred by CCM.
4. The core will collect sperm and cryopreserve in 9 straws (fewer straws if there is only one male).
5. Quality assessment will be performed via IVF using sperm from 1 straw. The fertilization rate is calculated by the percentage of 2-cell stage embryos after overnight incubation.

The core will store the cryopreserved sperm in our liquid nitrogen tank up to one year. Investigators will need to coordinate the transfer of the cryo stock of their mouse lines to a commercial company for long-term storage

What is expected

1. Generally, 9 straws of cryopreserved sperm will be produced.
2. Quality control is completed for each mouse line preserved. A fertilization rate of 20% or above is a passing rate for our QC.
3. If the QC test does not pass our standard, the core offers to repeat the cryopreservation at 50% cost.
4. If a reproductive defect is known for the specific GEM model, the total number of straws produced, and sperm quality could be low. In such an instance, additional cryopreservation attempts will be done at 100% cost.
5. Males older than 7 months of age can be provided for sperm cryopreservation in extreme circumstances. Please contact the core if this needs to occur. However, if sperm number or quality is determined to be low, the core will not repeat the cryopreservation at a reduced cost.

Embryo cryopreservation is an efficient, reliable, and cost-effective means of safeguarding and maintaining stocks of critically GEM resources. The core will coordinate with Investigator by the mouse age to schedule the cryopreservation date. The core recommends freezing 150 to 500 embryos for each line of mice. This number usually requires multiple rounds of producing embryos for freezing.

What Will Happen

1. Investigator will submit a request form through iLab, filling out detail information on male and female mouse numbers and ages, and any known reproductive defects. An account and IACUC protocol number must be provided at this time. The Investigator IACUC protocol must be approved for all procedures performed in the Investigator laboratory.
2. The core will review investigator provided information and approve the request. The core will schedule the cryopreservation according to the appropriate mouse age and coordinate with the investigator.
3. Investigators will complete the following steps to prepare embryos:
   • a. Investigator will prepare 8-10 adult males and 8-10 young female mice at age of 3-5 weeks with appropriate genotypes.
   • b. One week before the scheduled cryopreservation date, pick up 1 tube of M2 media, 1 tube of PMSG, and 1 tube of HCG from the GERM Core. Note: Bring an ice bucket to put the hormones in and put them in a -20C freezer when you get back to your lab. Dilute the hormones right before you go to give the injections.
   • c. PMSG is administered via intraperitoneal injection (IP), at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for rederivation on Friday). Dilute the PMSG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
   • d. HCG is administered via IP injection 47 hours after PMSG, at 9 a.m. one day before the scheduled rederivation date (e.g. Thursday for procedure on Friday). Dilute the HCG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
   • e. Place the females with the males immediately after administering HCG.
   • f. The morning following the HCG injections, the females are removed from the male cages and oviducts are collected into M2 media provided by the GERM Core.
   • g. Label the tubes with the iLab request number. Deliver the oviducts to ABBR R119A and leave in the biosafety hood at room temperature no later than 9 a.m.
4. The core will dissect, digest and collect all oocytes from the oviducts and cryopreserve all fertilized 1-cell embryos.
5. The core will store the cryopreserved embryos in our liquid nitrogen tank up to one year. The Investigator will need to send the cryo stock of their mouse line(s) to a commercial company for long-term storage.

What is Expected

1. The number of embryos for cryopreservation depends on the number of embryos collected from investigator-delivered oviducts. We freeze all embryos at 30-40 embryos per straw.
2. Quality control for the freezing process is completed by thawing a batch of wild-type cryopreserved embryos processed alongside the investigator request on the same day, and visually checked to assess viability rate. A rate of over 60% viability passes our QC.
3. If there are no fertilized embryos collected, the core will charge 40% of cost. Investigators can repeat the cryopreservation at 100% cost.
4. If a reproductive defect is known for the specific GEM model, the total number of embryos collected could be low. In such an instance, additional cryopreservation attempts will be done at 100% cost.

IVF service is used to rederive a mouse line imported from other institute, to rescue a mouse line that is in danger of being lost, to resuscitate a mouse line from previously cryopreserved sperm, or to facilitate mating for backcross/production. Live mice housed at BCM or cryopreserved sperm can be used as a source material.

What Will Happen

1. The Investigator will submit an IVF request form through iLab with detailed information about the sperm or mice. If cryopreserved sperm from an outside source is to be used, please have the IVF request in place prior to delivery of the sperm. the IACUC protocol number and account must be provided at this time. The Investigator’s IACUC protocol must be approved for all procedures performed in the Investigator’s laboratory.
2. Sperm or live mice delivery to be coordinated by the investigator.
   • a. If live mice are used, please submit a request to have the male mouse (mice) transferred by CCM or delivered by the Investigator to ABBR R119 by 8 am on the scheduled IVF day. For CCM transfers, please ark clearly on the transfer request that the mice are for IVF, must be transferred the same day as the scheduled request for IVF, and are required to be in place by 8 am. The core will recover sperm from the male for the IVF procedure. The male(s) will be euthanized and cannot be returned to the Investigator.
   • b. If cryopreserved sperm is used, the core will schedule the IVF procedure after the sperm arrives. The investigator can have the sperm shipped to their own lab and hand-deliver to the GERM Core or they can have the sperm shipped directly to the core with prior communication.
3. The core will perform IVF on wild-type oocytes from C57BL/6J, C57BL/6N, FVB or ICR mice. 15 female mice will be used for the IVF with superovulation. 300-500 oocytes are expected to be able to collect for the IVF.
4. After overnight culture, all 2-cell embryos will be transferred to ICR pseudo pregnant females to carry gestation to term in core.
5. Live-born founder animals will be held by the GERM Core until 10-14 days of age and subsequently transferred to the Investigator. Investigators will perform genotyping to identify the desired founder mice.

What is Expected

1. The number of pups to be generated by IVF varies widely, from less than 10 to over 60 pups, with occasionally no pups born. Sperm quality directly correlates with the number of oocytes able to be fertilized, and subsequently the number of pups born.
2. If no pups are born, the core offers to repeat the IVF at 50% cost if there is another male mouse or straw of sperm available.
3. Please contact the core if use of inbred strains other than C57BL/6J, C57BL/6N, FVB, or ICR are desired.

The goal of this service is to provide investigators with age-matched GEM models for their experiments. The complexity of current GEM models requires the maintenance of large breeding colonies and considerable time to acquire the appropriate number of mice for experiments. The GERM Core will utilize superovulation and embryo transfer to provide Investigators with the required numbers of mice of specific genotypes necessary for their experiments.

What Will Happen

1. Investigator will submit a request form through iLabs, filling out detail information on male and female mouse numbers and ages. An account and IACUC number must be provided at this time. The Investigator IACUC protocol must be approved for all procedures performed in the Investigator laboratory.
2. The core will review investigator-provided information and approve the request. The core will schedule the colony expansion according to the female mouse age and coordinate with the investigator.
3. Investigators will complete the following steps to prepare embryos:
   • a. Investigator will prepare 8-10 adult males and 8-10 young female mice at age of 3-5 weeks with appropriate genotypes.
   • b. One week before the scheduled service date, pick up 1 tube of M2 media, 1 tube of PMSG, and 1 tube of HCG from the GERM Core. Note: Bring an ice bucket to put the hormones in and put them in a -20C freezer when you get back to your lab. Dilute the hormones right before you go to give the injections.
   • c. PMSG is administered via intraperitoneal injection (IP), at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for rederivation on Friday). Dilute the PMSG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
   • d. HCG is administered via IP injection 47 hours after PMSG, at 9 a.m. one day before the scheduled rederivation date (e.g. Thursday for procedure on Friday). Dilute the HCG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
   • e. Place the females with the males immediately after administering HCG.
   • f. The morning following the HCG injections, the females are removed from the male cages and oviducts are collected into M2 media provided by the GERM Core.
   • g. Label the tubes with the iLab request number. Deliver the oviducts to ABBR R119A and leave in the biosafety hood at room temperature no later than 9 a.m.
4. The core will dissect, digest, and collect all oocytes from the oviducts, identify and transfer fertilized 1-cell embryos to pseudo pregnant ICR females to allow gestation.
5. Live-born founder animals will be held by the GERM Core until 10-14 days of age and subsequently transferred to the Investigator.

What is Expected

1. The GERM core will transfer about 150 embryos if available to 10 of 0.5-day pseudo pregnant recipient mice.
2. With good superovulation, it is expected that the Investigator will likely receive30-40 mice for experimental purposes.
3. If there are no fertilized embryos collected, the core will charge 40% of cost. Investigators can repeat the colony expansion at 100% cost.

The integrity of a GEM facility is dependent upon the ability to maintain the mouse colony in a specific pathogen free (SPF) state. Therefore, when BCM investigators desire to import mouse strains from facilities that are not SPF or when an outbreak of contamination occurs in a mouse colony, the GERM Core will help investigators to rederive their mouse strains using embryo transfer to recipient SPF female mice.

What Will Happen

1. Investigator will submit a request form through iLab, filling out information on the number and age of male and female mice to be used. An account number and IACUC protocol number must be provided at this time. The investigator’s IACUC protocol must be approved for all procedures performed in the Investigator laboratory.
2. The core will review the investigator provided information and approve the request. The core will schedule the mouse strain rederivation according to the female mouse age and coordinate with the investigator or CCM if the mice are in quarantine.
3. Investigators will complete the following steps to prepare embryos:
   • a. Investigator prepares 2+ adult males and 2-4+ young female mice at 3.5-5 weeks of age and of the appropriate genotypes.
   • b. One week before the scheduled rederivation date, pick up 1 tube of M2 media, 1 tube of PMSG, and 1 tube of HCG from the GERM Core (Location for pick up: N630.01 in Alkek Bldg.). Note: Bring an ice bucket to put the hormones in and put them in a -20C freezer when you get back to your lab. Dilute the hormones right before you go to give the injections.
   • c. PMSG is administered via intraperitoneal injection (IP), at 10 a.m. three days before the scheduled rederivation date (e.g. Tuesday for rederivation on Friday). Dilute the PMSG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
   • d. HCG is administered via IP injection 47 hours after PMSG, at 9 a.m. one day before the scheduled rederivation date (e.g. Thursday for rederivation on Friday). Dilute the HCG with 950 ul of 0.9% saline and administer 0.1cc IP per mouse.
   • e. Place the females with the males immediately after administering HCG.
   • f. The morning following the HCG injections, the females are removed from the male cages and oviducts are collected into M2 media provided by the GERM Core.
   • g. Label the tubes with the iLab request number. Deliver the oviducts to ABBR R119A and leave in the biosafety hood at room temperature no later than 9 a.m.
4. The core will dissect, digest, and collect all oocytes from the oviducts, identify and transfer all fertilized 1-cell embryos to pseudo pregnant recipient ICR female mice to allow gestation.
5. Live-born founder animals will be held by the GERM Core until 10-14 days of age and subsequently transferred to the investigator.

What is Expected

The GERM core will transfer all 1-cell embryos to 0.5-day pseudo pregnant recipient mice.

The number of expected live-born offspring will vary and will be dependent on the number of embryos transferred. On average, 15-20% of transferred embryos result in live-born offspring.

If there are no fertilized embryos collected, the core will charge 40% of cost. Investigators can repeat the rederivation at 100% cost.

The GERM Core will rent out dry shippers for cryopreserved sperm and embryo shipment. Please request a dry shipper rental through iLabs and contact the GERM Core with questions about dry shipper use.

What Will Happen

1. Investigator will submit a request form through iLab, filling out information as requested.
2. The core will approve the request and propose a charge based on the estimated days for rental.
3. Please note that there will be a $2000 deposit for the dry shipper in case it gets lost or damaged. This charge will be removed when the core receives the dry shipper back in good condition.
4. The core will coordinate with the investigator on which day to pick up the dry shipper from N630 based on the core dry shipper’s availability. The investigator will return the dry shipper immediately once it is received back to BCM.

Traditional transgenes are defined here as transgenes cloned into standard bacterial vectors. These transgene constructions, not including the vector, are usually less than 10 kb. These constructions are usually coding regions fused to promoter elements. If you are attempting to express cDNA sequences, it would be helpful to include an intron and a good polyadenylation signal. The design of these transgenic constructs should include a means to isolate the transgene from the plasmid backbone. The purity of the transgene fragment to be injected is critical for efficient generation of transgenic mice. In order to ensure that the transgene fragment is "Microinjection Quality", the GERM Core will isolate the transgene unless the investigator has a specific reason to isolate the fragment themselves.

What Will Happen

1.  Investigator will submit a request form through iLabs. An account and IACUC protocol number must be provided at this time.
2. Th investigator will generate their targeting construct. The GERM Core does not provide construct generation as a service. However, we will review the overall design if requested. The investigator is asked to follow the protocol below for sample submission:
   • Digest 100 ug of plasmid with the appropriate restriction enzyme(s) in a 300 ul reaction volume.
   • Run an aliquot of the digest on an agarose gel and photograph the results.
   • Indicate on the photograph the band to be isolated along with the size of the fragment.
   • Attach photograph and Eppendorf tube containing the rest of the digest to a print-out request form and deliver to the GERM Core (Delivery location: N630.01 in Alkek bldg.).
   • Note: The transgene must clearly separate from the plasmid backbone during electrophoresis. For example: If the backbone is 2.9 kb and the transgene is 3.0 kb, they will not separate on a normal gel. Additional enzymes would need to be used to allow at least a 1 kb difference between the fragments. If you can't digest the backbone free from the transgene, you may have to redesign.
3. The GERM core will use agarose gel electrophoresis to isolate the desired DNA fragment.
4. The Core will perform DNA purification, quantification, QC, and preparation of high-quality DNA in microinjection buffer.
5. The core will prepare and collect 1-cell embryos from C57BL/6J, C57BL/6N, or FVB/NJ after superovulation and perform microinjections of 150-200 embryos. The surviving embryos will be transferred to pseudo pregnant females.
6. Live-born founder animals will be held by the GERM Core until 10-14 days of age and subsequently transferred to the Investigator.
7. Investigator will genotype and identify the successfully targeted founder mice and subsequently mate them to identify germline transmission.

What is Expected

1. The number of live-born pups can significantly vary depending on the transgenic construct with very few to 50+ pups born.
2. A minimum of 5 live-born offspring is guaranteed. If 5 live-born offspring are not produced, the microinjection will be repeated.
3. Due to random integration, the expression pattern and levels of a transgene can significantly very from founder to founder. Additionally, random integration can alter the expression of an endogenous gene, causing phenotypes not directly attributable to the transgene itself. Therefore, independent lines should be derived from several founder animals and screened for expected transgene expression levels and patterns and expected phenotypes. Generally, a phenotype associated with the transgene, and not a disrupted endogenous gene, should be observable in multiple founder lines.
4. Founder animals can be mosaic and may carry several integration sites that can be independently transmitted to the next generation. Variability in phenotype and transgene expression level may be due to various sites segregating within a transgenic line.

If large fragments of genomic DNA averaging 100-300 kb need to be microinjected, then Bacterial Artificial Chromosome (BAC) clones harboring large genomic DNA fragments (100-300 Kb) can allow faithful tissue specific expression in a transgenic setting. However, due  to  their  size, the amount  of DNA that can be microinjected into an embryo is significantly less than a standard transgene and the constructs can easily shear, reducing efficiency of generating transgenic mice. Integrated copy number is also typically less than a standard transgenic construct. Successful generation of BAC transgenics is highly dependent upon the designing and preparation of the DNA. 

What Will Happen

1. Investigator will submit a request form through iLabs. An account and IACUC protocol number must be provided at this time.
2. The investigator is responsible for preparing BAC DNA (linearized or circular) for microinjection. The GERM Core does not provide preparation services; however recommended protocols and kit suggestions will be provided. It is critical that a kit designed for large constructs is used to minimize shearing.
3. The investigator is also responsible for quantifying the BAC DNA preparation. DNA must meet the following parameters:
   • 260/280 of 1.70 to 2.0
   • Digestion of an aliquot of BAC DNA with a frequent cutter, e.g. BamHI, and run on a 0.8% agarose gel. A distinct, predicted banding pattern based on the BAC sequence (a BAC fingerprint) should be visible.
   • If linearized, run DNA on a pulse field gel. You should see a single, sharp band of the expected size.
   • Do not freeze BAC DNA as it will shear after thawing.
4. Investigator will deliver the BAC DNA prepared to the GERM Core with a copy of the iLab request form attached 1-7 days prior to the scheduled microinjection. BAC DNA will degrade/shear more rapidly than a standard plasmid.
5. The GERM core will store the resuspended DNA at 4ºC until the microinjection day.
6. On the morning of microinjection, the GERM core will repeat the 260/280 measurements and dilute it to 1ng/ul into microinjection buffer.
7. The core will prepare and collect 1-cell embryos from C57BL/6J, C57BL/6N, or FVB/NJ after superovulation and perform microinjections to 150-200 embryos. The survival embryos will be transferred to pseudo pregnant females.
8. Live-born founder animals will be held by the GERM Core until 10-14 days of age then transferred to the User Investigator.
9. Investigator will genotype and identify the successfully targeted founder mice and subsequently mate the founders to identify germline transmission.

What is Expected

1. The number of live-born pups can significantly vary depending on the transgenic construct with very few to 50+ pups born.
2. A minimum of 5 live-born offspring is guaranteed. If 5 live-born offspring are not produced the microinjection will be repeated.
3. Due to random integration, the expression pattern and levels of a transgene can significantly very from founder to founder. Additionally, random integration can alter the expression of an endogenous gene, causing phenotypes not directly attributable to the transgene itself. Therefore, independent lines should be derived from several founder animals and screened for expected transgene expression levels and patterns and expected phenotypes. Generally, a phenotype associated with the transgene, and not a disrupted endogenous gene, should be observable in multiple founder lines.
4. Founder animals can be mosaic and may carry several integration sites that can be independently transmitted to the next generation. Variability in phenotype and transgene expression level may be due to various sites segregating within a transgenic line.
5. BAC DNA can easily shear and only fragments of the BAC DNA may integrate into the genome. If a BAC harboring genomic DNA from one inbred strain (e.g. 129) is microinjected into another strain (e.g. FVB) PCR-based screening of microsatellite markers or SNP variant screening can be used to identify regions of BAC DNA integrated. If a BAC harboring genomic DNA from human is used, PCR-based screening for human sequences, Sanger sequencing for human sequences, or Southern blotting for human repeat sequences (e.g. Alu elements) can be used to identify regions of BAC DNA integrated.

Investigator submits a targeting strategy and demonstrates a proven screening protocol. After approval of investigator’s approach, the core will initiate electroporation in mouse ES cells. Investigator-screened targeted clones will be expanded by the core, sent for chromosome counts, and microinjected into blastocysts for chimera production. Investigators will receive potential chimeras and breed to germline transmission.  All projects are scheduled through iLab on a first come first serve basis. 

For 129 ES cells, C57BL/6J blastocysts generated via super ovulation will be used to generate chimera founder mice; for C57BL/6N ES cells, FVB 8-cell embryos generated via super ovulation will be used to generate chimera founder mice.

What Will Happen

1. The investigator will submit a request form through iLab with a detailed gene targeting strategy and proven screening protocol. An account and IACUC number must be provided at this time. The ES cell line to be used is selected at this time. ES cell lines available for targeting are:
   • AB2.2: 129/SvEvBrd [Hprtb-m2]
   • E14Tg2a: 129/OlaHsd [Hprtb-m3] (feeder free)
   • JM8.F6: C57BL6/Ntac
   • JM8.N4: C57BL6/Ntac
2. The core will review investigator’s targeting strategy and approve project or make recommendations.
3. The investigator will generate their targeting construct. The GERM Core does not provide construct generation as a service.
4. The core will schedule the project and prepare cells for electroporation.
5. The investigator will prepare linearized or circular vector for electroporation and bring to the core as pellet in 100% ethanol.  Projects involving B6 ES cells require 250ug of DNA.  Projects involving 129 ES cells require 150ug of DNA.
6. The core will prepare mouse ES cells for electroporation, perform electroporation, and select/isolate clones. The core will pick up to three 96 well plates of clones. Additional plates will incur extra cost. Triplicates of each plate will be generated: two copies of each plate will be prepared as lysate and given to investigator for targeting screen; one copy will be stored by the core at -80C for expansion.
7. Investigators will perform all ES cell genotyping and must return results to the core no later than three months after receiving lysate plates. The GERM Core does not provide ES cell genotyping services.
8. The Core will expand up to 10 targeted clones. Clones will be graded according to culture morphology in order to select three clones to send out for chromosome counts and finally microinjection. ES cell clones must have a minimum of 50% euploid nuclei for microinjections.
9. The Core will provide investigator a DNA pellet for confirmation of expanded clone targeting; the investigator will notify that the expanded clones harbor a targeted allele.
10. The core will schedule microinjection of ES cells into blastocysts/8-cell embryos after chromosome counts are determined. The core will use appropriate recipient embryos for coat color identification of chimeras and offspring. Examples:
    • 129 (agouti) ES cells: B6 embryos
    • B6 (agouti) ES cells: FVB 8-cell embryos
    • B6 (black/non-agouti) ES cells: FVB 8-cell embryos
11. Superovulation (129 and B6 ES cells) will be used to generate embryos for microinjection.
12. The core will microinject 45 embryos per clone.  
    • The core will microinject up to 2 clones for 129-derived and up to 3 clones for B6-derived ES cells.
    • Additional clones will incur extra cost.
13. Dams will be allowed to litter in core space. Litters will be transferred to investigator space at two weeks of age. The number, sex, and percent ES cell coat color contribution of chimeras will be indicated.
14. Investigator will breed chimera and confirm germline transmission of gene.

 What is Expected

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • Freezing of 96 well plates of picked ES cell clones
   • Expansion and freezing of selected ES cell clones
   • ES cell chromosome counting
   • Blastocyst injection
2. If targeted ES cell clones are not identified, the electroporation will be repeated at 50% cost.
3. Chimeric animals with coat color contribution from the injected ES cell clones ranging from 10%-100%. Coat color contributions does not necessarily represent ES cell contribution to cell lineages in all tissues, in particular the germline. However, males with the highest amount of coat color contribution from the ES cells should be prioritized for breeding and as many males as possible should be bred.
4. The number of chimeric animals produced and the range of coat color contribution from an ES cell clone can vary widely and can depend on the pluripotent potential of the ES cell clone and potentially the targeted gene.
5. Germline transmission of the targeted allele can require several rounds of breeding attempts. Transmission efficiency is dependent on overall ES cell contribution to the germ cell population (influenced by chromosome stability and pluripotent capacity). Good coat color contribution is not a guarantee of germline transmission. Moreover, as targeting in ES cells almost always results in only one modified allele (heterozygosity), coat color and not the targeted allele can be transmitted. Targeted gene function can affect allele transmission.
6. Female chimeras should not be bred. They will often be sterile and male offspring will be sterile.
7. There is no guarantee that chimeric mice will be produced or that chimeras will breed. Additional microinjections with the same ES cell clones or different clones will be done at 100% of cost.

Investigator submits a targeting strategy and demonstrates a proven screening protocol.  After approval of investigator’s approach the core will initiate electroporation in mouse ES cells. Investigator-screened targeted clones will be expanded by the core, sent for chromosome counts, and microinjected into blastocysts for chimera production. Investigators will receive potential chimeras and breed to germline transmission.  All projects are scheduled through iLab on a first come first serve basis.

For 129 ES cells, C57BL/6J blastocysts generated via super ovulation will be used to generate chimera founder mice; for C57BL/6N ES cells, FVB 8-cell embryos generated via super ovulation will be used to generate chimera founder mice.

What will Happen

1. The investigator will submit a request form through iLab with a detailed gene targeting strategy and proven screening protocol. An account and IACUC number must be provided at this time. The ES cell line to be used is selected at this time. Choices are:
   • AB2.2: 129/SvEvBrd [Hprtb-m2]
   • E14Tg2a:  129/OlaHsd [Hprtb-m3] (feeder free)
   • JM8.F6: C57BL6/Ntac
   • JM8.N4: C57BL6/Ntac
2. The core will review investigator’s targeting strategy and approve project or make recommendations.
3. The investigator will generate their targeting construct. The GERM Core does not provide construct generation as a service.
4. The core will schedule the project and prepare cells for electroporation.
5. The investigator will prepare linearized or circular vector for electroporation and bring to the core as pellet in 100% ethanol. Projects involving B6 ES cells require 75ug of DNA. Projects involving 129 ES cells require 50ug of DNA.
6. The core will prepare mouse ES cells for electroporation, perform electroporation, and select/isolate clones. The core will pick up to 48 clones into a 96 well plate. Additional clones will incur extra cost. Triplicates of each plate will be generated: two copies of each plate will be prepared as lysate and given to investigator for targeting screen; one copy will be stored by the core at -80C for expansion.
7. Investigators will perform all ES cell genotyping and must return results to the core no later than three months after receiving lysate plates. The GERM Core does not provide ES cell genotyping services.
8. The Core will expand up to 10 targeted clones. Clones will be graded according to culture morphology in order to select three clones to send out for chromosome counts and finally microinjection. ES cell clones must have a minimum of 50% euploid nuclei for microinjections.
9. The Core will provide investigator a DNA pellet for confirmation of expanded clone targeting; the investigator will notify that the expanded clones harbor a targeted allele.
10. The core will schedule microinjection of ES cells into blastocysts/8-cell embryos after chromosome counts are determined. The core will use appropriate recipient embryos for coat color identification of chimeras and offspring. Examples:
    • 129 (agouti) ES cells: B6 embryos
    • B6 (agouti) ES cells: FVB 8-cell embryos
    • B6 (black/non-agouti) ES cells: FVB 8-cell embryos
11. Superovulation (129 and B6 ES cells) will be used to generate embryos for microinjection.
12. The core will microinject 45 embryos per clone.  
    • The core will perform microinjections of up to 2 clones for 129-derived and up to 3 clones for B6-derived ES cells.
    • Additional clones will incur extra cost.
13. Dams will be allowed to litter in core space. Litters will be transferred to investigator space at two weeks of age. The number, sex, and percent ES cell coat color contribution of chimeras will be indicated.
14. Investigator will breed chimera and confirm germline transmission of gene.

What is Expected

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • Freezing of 96 well plates of picked ES cell clones
   • Expansion and freezing of selected ES cell clones
   • ES cell karyotyping
   • Blastocyst injection
2. If targeted ES cell clones are not identified, the electroporation will be repeated at 50% cost.
3. Chimeric animals with coat color contribution from the injected ES cell clones ranging from 10%-100%. Coat color contributions does not necessarily represent ES cell contribution to cell lineages in all tissues, in particular the germline. However, males with the highest amount of coat color contribution from the ES cells should be prioritized for breeding and as many males as possible should be bred.
4. The number of chimeric animals produced and the range of coat color contribution from an ES cell clone can vary widely and can depend on the pluripotent potential of the ES cell clone and potentially the targeted gene.
5. Germline transmission of the targeted allele can require several rounds of breeding attempts. Transmission efficiency is dependent on overall ES cell contribution to the germ cell population (influenced by chromosome stability and pluripotent capacity). Good coat color contribution is not a guarantee of germline transmission. Moreover, as targeting in ES cells almost always results in only one modified allele (heterozygosity), coat color and not the targeted allele can be transmitted. Targeted gene function can affect allele transmission.
6. Female chimeras should not be bred. They will often be sterile and male offspring will be sterile.
7. There is no guarantee that chimeric mice will be produced or that chimeras will breed. Additional microinjections with the same ES cell clones or different clones will be done at 100% of cost.

The Core will assist investigators in expansion of mouse ES cells clones purchased from repositories, obtained from researchers at other institutions, or targeted in their own lab. We will expand cells and send sample for murine virus testing prior to injection. Microinjection will be processed, and investigators will receive potential chimeras and breed to germline transmission. For 129 ES cells, C57BL/6J blastocysts generated via super ovulation will be used to generate chimera founder mice; for C57BL/6N ES cells, FVB 8-cell embryos generated via super ovulation will be used to generate chimera founder mice.

What Will Happen

1. The investigator will submit a request form through iLab with a detailed gene targeting strategy and proven screening protocol. An account and IACUC number must be provided at this time.
2. Investigator will bring frozen vial(s) of ES cell clones to the core along with culture protocol and recipes.
3. The core will review investigator’s request and schedule a date to thaw clone.
4. The core will expand clone in the appropriate media and on feeder cells if needed, split into five vials, freeze and store in liquid nitrogen.
5. The core will submit ES cell sample to RADIL laboratories for murine virus testing. Virus testing is mandatory by the BCM Center for Comparative Medicine prior to ES cells being microinjected. If ES cells have also not been karyotyped, the Core can also coordinate cells to be sent out for chromosome counting. A cell pellet will be given to investigator should he/she need to do further genotyping of cells.
6. The core will schedule microinjection of cells into blastocysts/8-cell embryos after receiving murine virus test results. The core will use appropriate recipient embryos for coat color identification of chimeras and offspring. Examples:
   • 129 (agouti) ES cells: B6 embryos
   • B6 (agouti) ES cells: FVB 8-cell embryos
   • B6 (black/non-agouti) ES cells: FVB 8-cell embryos
7. Superovulation (129 and B6 ES cells) will be used to produce embryos.
8. The core will microinject 45 embryos per clone.  
   • The core will perform microinjections of up to 2 clones per request.
   • Additional clones will incur extra cost.
9. Dams will be allowed to litter in core space. Litters will be transferred to investigator space at two weeks of age. The number, sex, and percent ES cell coat color contribution of chimeras will be indicated.
10. Investigator will breed chimera and confirm germline transmission of gene.

What is Expected

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • ES cell expansion and freezing
   • ES cell virus testing
   • Blastocyst injections
2. Chimeric animals with coat color contribution from the injected ES cell clones ranging from 10%-100%. Coat color contributions does not necessarily represent ES cell contribution to cell lineages in all tissues, in particular the germline. However, males with the highest amount of coat color contribution from the ES cells should be prioritized for breeding and as many males as possible should be bred.
3. The number of chimeric animals produced and the range of coat color contribution from an ES cell clone can vary widely and can depend on the pluripotent potential of the ES cell clone and potentially the targeted gene.
4. Germline transmission of the targeted allele can require several rounds of breeding attempts. Transmission efficiency is dependent on overall ES cell contribution to the germ cell population (influenced by chromosome stability and pluripotent capacity). Good coat color contribution is not a guarantee of germline transmission. Moreover, as targeting in ES cells almost always results in only one modified allele (heterozygosity), coat color and not the targeted allele can be transmitted. Targeted gene function can affect allele transmission.
5. Female chimeras should not be bred. They will often be sterile and male offspring will be sterile.
6. There is no guarantee that chimeric mice will be produced or that chimeras will breed. Additional microinjections with the same ES cell clones or different clones will be done at 100% of cost.

The genome editing efficiency of up to 2 guide RNAs selected by the Core as part of a project design or 2 guide RNAs selected by an investigator and approved by the Core will be tested in mouse embryos.

What Will Happen

1. The Core will create guide testing requests after the User Investigator initiates a CRISPR-KO or CRISPR-KI project in iLab. The Core will design or review the investigator design for creating their desired allele. Guide testing is most useful for CRISPR-KI projects requiring precise placement of inserted sequence and the synthesis of a costly repair donor DNA. The Core will use the same account number provided for the initial CRISPR request for the guide testing request.
2. The Core will design a PCR-based assay to detect indels resulting from NHEJ. A PCR protocol will be optimized using wild-type DNA.
3. The Core will have the guide RNA synthesized by an approved vendor. Lab produced guide RNAs will not be tested.
4. The guide RNA will be checked for concentration and degradation after resuspension.
5. The guide RNA and Cas9 protein will be complexed into RNPs and an electroporation mix will be prepared.
6. 40-50 C57BL/6J embryos collected from superovulated females will be electroporated and subsequently cultured to the blastocyst stage. Embryos from other strains can be tested if necessary. Please contact the GERM Core to discuss the necessary steps involved to use other strains.
7. Blastocyst DNA will be isolated from single embryos or pooled samples of 3-5 embryos.
8. PCR will be performed on the samples and the resulting PCR products will be analyzed using a QIAxcel to visualize the presence of indels.
9. If no indels are identified from the QIAxcel analysis, the samples will be Sanger sequenced. ICE analysis (Synthego) will be used to deconvolute sequencing traces for each sample.
10. Guide efficiency will be determined by the percentage of samples with indel alleles when compared to WT PCR products. 

What to Expected

1. Some embryos will die during culture or enough DNA may not be isolated for PCR. We will guarantee a minimum of 12 blastocysts that produce a PCR product. Additional electroporations will be performed if necessary.
2. Genome editing in mouse zygotes often results in mosaicism. Thus, multiple indel alleles are likely to be detected within a single blastocyst. Efficiency of the guide will be determined by the percentage of alleles with an indel.
3. To produce mouse lines a guide RNA should have the following efficiencies:
   • Projects requiring NHEJ (e.g. knockout allele production): At least 60% of blastocysts with a detected indel allele
   • Projects requiring HDR (e.g. point mutations): At least 75% of blastocysts with a detected indel allele. 

CRISPR/Cas9-mediated NHEJ in mouse embryos will be used to generate founder animals harboring insertion/deletion (indel) or interval deletion knockout alleles. At this time, the core can perform genome editing with S.p. Cas9 or A.s. Cas12a/Cpf1.

What Will Happen

1. The investigator will initiate a project in iLab. An IACUC protocol number, mouse transfer information, and a charge account must be provided at this time. CRISPR projects can only be paid for using a BCM-associated account.
2. A consultation meeting between the core and investigator will be held to discuss the desired genome modification.
3. Based on this discussion the core will:
   • Design a genome editing approach to produce and detect the desired allele, including selection of a critical exon, selection of guide RNAs, and a PCR-based genotyping scheme. The design will be reviewed with the investigator and modifications made if necessary.
   • Review genome editing approaches designed by the investigator, including verification that the approach will produce a null allele, independent computational assessment of guide RNA quality, and review of the genotyping approach. The requested information must be provided to the core and the core must approve the approach prior to proceeding to the next steps.
4. The core will have guide RNA(s) synthesized by an approved vendor. Lab produced guide RNAs will not be used.
5. Guide RNA(s) will be checked for concentration and degradation after resuspension.
6. Investigators can opt to have the guide RNAs tested for genome editing efficiency in mouse zygotes prior to the production attempt [see Guide RNA testing in mouse zygotes].
7. The guide RNA(s) and Cas9 protein will be complexed into RNPs and an electroporation mix in nuclease-free buffer will be prepared at concentrations determined by the core.
8. 80 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be electroporated and transferred to pseudopregnant females. Please contact the GERM core to discuss the necessary steps involved to use other strains.
9. Live-born founder animals will be held by the GERM core until 14 days of age and subsequently transferred to the investigator.
10. Genotyping:
    • It is recommended that the core perform genotyping of live-born offspring for the desired genome editing event [view Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. This service will be done at an additional cost.
    • If the investigator conducts their own genotyping, core Staff can review genotyping results at an additional cost.
11. If the core performs genotyping, they will report back to the investigator the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If the investigator conducts their own genotyping, the same information will be reported back to the core. 

What to Expected

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • Design completion or verification
   • Reagent ordering
   • Embryo electroporation
2. A minimum of 10 live-born offspring. If 10 live-born offspring are not produced, the electroporation will be repeated for 80 embryos.
3. At least one live-born animal with the desired genome editing (one founder animal).
4. If one founder is not identified:
   • If the guide RNAs were (i) tested in mouse embryos by the core and the core approved their use based on the genotyping results and (ii) the core conducted the founder genotyping, the core can review results, redesign the approach and reagents if needed, and the electroporation can be repeated. Project costs associated with re-design and additional embryo electroporation will be reduced by 50%. Costs associated with purchase of new guide RNAs and donor DNAs will not be reduced.
   • If the guide RNAs were not tested in mouse embryos or the investigator conducts their own genotyping, there will be no reduced costs for additional work.
5. Indel mutations are often too small to detect by conventional PCR. Sanger sequencing and trace analysis will need to be performed to identify and characterize alleles produced.
6. Exon deletion alleles are typically sufficient in size to detect by conventional PCR. The size of the interval deletion often varies between animals because NHEJ repair is semi-random. Sanger sequencing of deletion allele PCR products is necessary to verify the deletion and sequence at the NHEJ repair site.
7. Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at this stage. Moreover, the various alleles found in a founder can be passed onto the next generation. Thus, when breeding founders, the resulting N1 offspring must be PCR genotyped and Sanger sequenced to assess which alleles were inherited by which animals. A colony should be established from N1 animals harboring the same NHEJ allele (i.e. the same sequence at the repair junction). We recommend that N1 animals be backcrossed to wild-type animals. Intercrossing N1 animals is not recommended. 

CRISPR/Cas9-initiated HDR in mouse embryos will be used to generate founder animals harboring inserted sequence (point mutations, epitope tags, loxP sites) or specific interval deletion alleles. At this time, the core can perform genome editing with S.p. Cas9 or A.s. Cas12a/Cpf1. The targeting will be done in embryos using electroporation to introduce the CRISPR reagents. 

What Will Happen

1. The investigator will initiate a project in iLab. An IACUC protocol number, mouse transfer information, and a charge account must be provided at this time. CRISPR projects can only be paid for using a BCM-associated account.
2. A consultation meeting between the core and investigator will be held to discuss the desired genome modification.  
3. Based on this discussion the core will:
   • Design a genome editing approach to produce and detect the desired allele:
     • Point mutation alleles: With information from the investigator describing the desired point mutation, the core will select guide RNAs, design a suitable ssODN repair template, and design a PCR-based genotyping scheme. The donor design will include silent mutations to modify the target sequence in the donor DNA, and possibly introduce a novel restriction site to facilitate genotyping. The overall design will be reviewed with the Investigator and modifications made if necessary.
     • Epitope tag alleles: With information from the investigator describing the location of the epitope tag, the core will select guide RNAs, design an ssODN repair template to insert the specified tag, and design a PCR-based genotyping scheme. The design will be reviewed with the Investigator and modifications made if necessary.
     • Conditional alleles: The core will select a critical exon, select guide RNAs, design two ssODN repair templates to insert the loxP sequences, and design a PCR-based genotyping scheme. The design will be reviewed with the investigator and modifications made if necessary.
   • Review genome editing approaches designed by the investigator, including verification that the approach will produce the desired allele, independent computational assessment of guide RNA quality, and review of the genotyping approach. The requested information must be provided to the core and the core must approve the approach prior to proceeding to the next steps.
4. The core will have the guide RNA(s) synthesized by an approved vendor. Lab produced guide RNAs will not be used.
5. Guide RNA(s) will be checked for concentration and degradation after resuspension.
6. Investigators can opt to have the guide RNAs tested for genome editing efficiency in mouse zygotes prior to the production attempt [see Guide RNA testing in mouse zygotes].
7. The core will have the ssODN repair template(s) synthesized as custom Ultramers by IDT.
8. The guide RNA(s) and Cas9 protein will be complexed into RNPs and an electroporation mix will be prepared with the ssODN repair template(s) in nuclease-free buffer at concentrations determined by the core.
9. 200 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be electroporated and transferred to pseudopregnant females. Please contact the GERM core to discuss the necessary steps to use other strains.
10. Live-born founder animals will be held by the GERM core until 14 days of age and subsequently transferred to the investigator.
11. Genotyping:
    • It is recommended that the core perform genotyping of live-born offspring for the desired genome editing event [view Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. This service will be done at additional costs.
    • If the investigator conducts their own genotyping, core staff can review genotyping results at an additional cost.
12. If the core performs genotyping, they will report back to the investigator the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If the investigator conducts their own genotyping, the same information will be reported back to the core.

What to Expect

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • Design completion or verification
   • Reagent ordering
   • Embryo electroporation
2. A minimum of 5 live-born offspring. If less than 5 live-born offspring are produced, the electroporation will be repeated for an additional 100 embryos.
3. At least one live-born animal with the desired genome editing event (one founder animal).
4. If one founder is not identified:
   • If the guide RNAs were (i) tested in mouse embryos by the core and the core approved their use based on the genotyping results and (ii) the core conducted the founder genotyping, the core can review results, redesign the approach and reagents if needed, and the electroporation can be repeated. Project costs associated with re-design and additional embryo electroporation will be reduced by 50%. Costs associated with purchase of new guide RNAs and donor DNAs will not be reduced.
   • If the guide RNAs were not tested in mouse embryos or the investigator conducts their own genotyping, there will be no reduced costs for additional work.
5. Depending on the desired allele, conventional PCR is generally sufficient to detect evidence of genome editing:
   • a. Point mutation alleles: Typically, there will be almost no size difference between the edited and unedited allele. However, the inclusion of a novel restriction site in the repair template (introduced by silent mutation) can greatly facilitate detecting targeted animals. Sanger sequencing of PCR products from the desired allele is necessary to verify the sequence at the target site.
   • b. Epitope tag and conditional KO alleles: The inserted sequence in a correctly targeted allele will create a shift in the size of a PCR product between the edited and unedited allele. Sanger sequencing of PCR products from the desired allele is necessary to confirm the correct sequence at the target site.
6. Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at this stage. Moreover, the various alleles found in a founder can be passed onto the next generation. Thus, when breeding founders, the resulting N1 offspring must be PCR genotyped and Sanger sequenced to assess which alleles were inherited by which animals. A colony should be established from N1 animals harboring the same sequence confirmed HDR allele. We recommend that N1 animals be backcrossed to wild-type animals. Intercrossing N1 animals is not recommended. 

CRISPR/Cas-initiated HDR in mouse embryos will be used to generate founder animals harboring inserted sequence (loxP sites, fluorescent reporters, or other complex alleles) or specific interval deletion alleles. At this time, the core can perform genome editing with S.p. Cas9 or A.s. Cas12a/Cpf1. The targeting will be done in embryos using microinjection to introduce the CRISPR reagents.

What Will Happen

1. The investigator will initiate a CRISPR-KI request in iLab. The request form requires the gene name and a brief allele description. An IACUC protocol number, mouse transfer information, and a charge account must be provided at this time. CRISPR projects can only be paid for using a BCM-associated account.
2. A consultation meeting between the core and the investigator will be held to discuss the desired genome modification.
3. Based on this discussion the core will:
   • Design a genome editing approach to produce and detect the desired allele:
     • Conditional alleles: The core will select a critical exon, select guide RNAs, design (a) suitable repair template(s) to insert both LoxP sequences, and design a PCR-based genotyping scheme. The design will be reviewed with the investigator and modifications made if necessary.
     • Reporter and Recombinase KI alleles: With information from the investigator describing the location of the desired KI, the core will select guide RNAs, design a suitable repair template to insert the desired sequence, and design a PCR-based genotyping scheme. The design will be reviewed with the Investigator and modifications made if necessary.
     • Complex alleles: With information from the investigator describing the desired allele, the core will select guide RNAs, design a suitable repair template to insert the specified sequence, and design a PCR-based genotyping scheme. The core has the right to decline producing a design for any overly complicated allele that may be difficult to target in mouse zygotes using CRISPR reagents. Furthermore, the core may suggest that certain complex projects be attempted in ES cells through traditional targeting.
   • Review genome editing approaches designed by the investigator, including verification that the approach will produce the desired allele, independent computational assessment of guide RNA quality, and review of the genotyping approach. The requested information must be provided to the core and the core must approve the approach prior to proceeding to the next steps.
4. The core will have the guide RNA(s) synthesized by an approved vendor. Lab produced guide RNAs will not be used.
5. Guide RNA(s) will be checked for concentration and degradation after resuspension.
6. Investigators can opt to have the guide RNAs tested for genome editing efficiency in mouse zygotes prior to the production attempt [view Guide RNA testing in mouse zygotes]. This service is strongly recommended for precise genome edits requiring costly repair donor templates.
7. The core will provide a quote for the synthesis of the repair template, so that the investigator may have a PO generated for the purchase of the donor DNA.
8. Depending on the location of the microinjection, the core will assemble an appropriate microinjection mix:
   • For cytoplasmic injections the mix will contain the guide RNA(s), DNA repair template(s), and Cas9 mRNA in nuclease-free buffer at concentrations determined by the core.
   • For pronuclear microinjections (projects with dsDNA repair templates) the guide RNA(s) and Cas9 protein will be complexed into RNPs and the microinjection mix will be prepared with the repair template in nuclease-free buffer at concentrations determined by the core.
9. 200 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be microinjected and transferred to pseudopregnant females. Please contact the GERM core to discuss the necessary steps if other strains are desired.
10. Live-born founder animals will be held by the GERM core until 14 days of age and subsequently transferred to the investigator.
11. Genotyping:
    • It is recommended that the core perform genotyping of live-born offspring for the desired genome editing event [view Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. This service will be done at an additional cost.
    • If the investigator conducts their own genotyping, core staff can review genotyping results at an additional cost.
12. If the core performs genotyping, they will report back to the investigator the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If the investigator conducts their own genotyping, the same information will be reported back to the core.

What to Expect

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • Design completion or verification
   • Reagent ordering
   • Embryo microinjection
2. A minimum of 5 offspring transferred to the investigator’s colony. If less than 5 offspring are transferred, the microinjection will be repeated for an additional 100 embryos with a new preparation of the donor DNA.
3. At least one live-born animal with the desired genome editing event (one founder animal).
4. If one founder is not identified:
   • If the guide RNAs were (i) tested in mouse embryos by the core and the core approved their use based on the genotyping results and (ii) the core conducted the founder genotyping, the core will review the results to troubleshoot any potential limitations. A repeat microinjection may be the best approach for obtaining a founder. If a redesign is needed, project costs associated with re-design and additional embryo microinjections will be reduced by 50%. For reagent purchases on project redesigns the core will cover the cost of new gRNA(s), but the investigator will be responsible for the cost associated with purchase of the new donor DNA(s).
   • If the guide RNAs were not tested in mouse embryos or the investigator conducts their own genotyping, there will be no reduced costs for additional work.
5. Depending on the desired allele, conventional PCR is generally sufficient to detect evidence of genome editing. Genotyping schemes will be designed to only amplify PCR products in the correctly targeted allele. Additionally, the inserted sequence in a correctly targeted allele will create a shift in the size of a PCR product between the edited and unedited allele. Sanger sequencing of PCR products from the desired allele is necessary to confirm the correct sequence at the target site.
6. Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at this stage. Moreover, the various alleles found in a founder can be passed onto the next generation. Thus, when breeding founders, the resulting N1 offspring must be PCR genotyped and Sanger sequenced to assess which alleles were inherited by which animals. A colony should be established from N1 animals harboring the same sequence confirmed HDR allele. We recommend that N1 animals be backcrossed to wild-type animals. Intercrossing N1 animals is not recommended.

CRISPR/Cas-initiated HR in mouse embryos will be used to generate founder animals harboring inserted sequence targeted to the ROSA26 locus. The targeting will be done in embryos using microinjection to introduce the CRISPR reagents. The investigator will provide their own targeting donor developed from one of the vectors available from the Kühn Lab. Vectors available can be found on Addgene:

• www.addgene.org/Ralf_Kuehn/
• Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.BMC Biotechnol. 2016 Jan 16;16(1):4. doi: 10.1186/s12896-016-0234-4.

Refer to Fig. 7 for the constructs that can be used.

The individual investigators will do all donor design and production. For additional resources to assist in donor production, contact the core. It is recommended that the investigator request this service once they have a suitable donor DNA ready for microinjection.

What Will Happen

1. The investigator will initiate a CRISPR-KI request in iLab. The investigator will list the gene name as Rosa26; specify that the investigator created the design; and select from a drop down list of allele types “Rosa26 locus KI”. An IACUC protocol number, mouse transfer information and a charge account must be provided at this time. CRISPR projects can only be paid for using a BCM-associated account.
2. A consultation meeting between the core and investigator will be held to verify the standard components described by Chu et al (see reference above) are to be used.
3. The core will provide the published guideRNA. Lab produced guide RNAs will not be used.
4. The investigator will provide 6-8 ug of donor DNA for phenol: chloro form extraction by the core, prior to assembling the microinjection mix.
5. The core will assemble an appropriate microinjection mix for pronuclear microinjections: the guide RNA and Cas9 protein will be complexed into RNPs and the repair template added subsequently, in nuclease-free buffer.
6. 200 C57BL/6J, C57BL/6N, or FVB/NJ embryos collected from superovulated females will be electroporated and transferred to pseudopregnant females. Contact the GERM core to discuss the necessary steps if other strains are desired.
7. Live-born founder animals will be held by the GERM core until 14 days of age and subsequently transferred to the investigator.
8. Genotyping:
   • It is highly recommended that the core perform genotyping of live-born offspring for the correct targeting at the ROSA26 locus[see Founder and N1 animal PCR genotyping Service; Founder and N1 Sanger sequencing Service]. The core has optimized long PCR strategies to verify correct targeting at each homology arm.
   • If the investigator conducts their own genotyping, core Staff can review genotyping results at an additional cost.
9. If the core performs genotyping, they will report back to the investigator the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If the investigator conducts their own genotyping, the same information will be reported back to the core.

What to Expect

1. Billing will occur in steps as project milestones are met. Billing will occur at the following steps:
   • Design verification
   • Embryo microinjection
2. A minimum of 5 offspring transferred to the investigator’s colony. If less than 5 offspring are transferred, the microinjection will be repeated for an additional 100 embryos with a new preparation of the donor DNA.
3. The core will not guarantee that at least one live-born animal with targeting at the Rosa26 locus is obtained. However, if genotyping is performed by the core, and no successfully targeted founders are identified, the microinjection will be repeated for another 100 embryos.
4. If the investigator conducts their own genotyping, the core will only repeat the microinjection at 100% cost.
5. For targeting at the Rosa26 locus, a long-range PCR reaction is needed to verify the correct insertion of the homology arms. Additionally, conventional PCR can be used to detect evidence of genome editing around the target site, and to screen for the KI sequence, however neither of these reactions will provide confirmation of a correctly targeted allele. The homology arm PCR products will only be produced in animals with a correctly targeted allele. Sanger sequencing of PCR products from the desired allele is necessary to confirm the correct sequence at the target site.
6. Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at this stage. Moreover, the various alleles found in a founder can be passed onto the next generation. Thus, when breeding founders, the resulting N1 offspring must be PCR genotyped to assess which animals inherited which alleles, and Sanger sequenced to confirm the targeted allele. A colony should be established from N1 animals harboring the same sequence confirmed HDR allele. We recommend that N1 animals be backcrossed to wild-type animals. Intercrossing N1 animals is not recommended.

General Description

CRISPR/Cas projects initiated with the core will be genotyped for desired genome editing events in founders using previously designed schemes or schemes approved by the core. For N1 animal genotyping, previously used genotyping designs for screening founders will be used to identify heterozygous animals inheriting the desired targeted allele. Tail clips, ear punches, or purified DNA samples can be submitted.

What Will Happen

1. The investigator will initiate a project in iLab. An account number must be provided at this time.
2. The investigator will specify the number of animals they have for genotyping and fill out the sample submission table. Samples should be labeled using the following convention: Use the 4-digit iLab request number (i.e. GERM-DL-2578) followed by a dash and a three digit number from 001 to the number of N samples submitted. For example, 2578-001, 2578-002, etc. Samples submitted without this naming convention will have these labels assigned to the samples accordingly.
3. The core will have oligonucleotides for PCR synthesized by an approved vendor.
4. The core will contact the investigator when it is ready to accept the samples. Please do not submit the samples until contacted by the core. Samples must be submitted in 1.5ml microfuge tubes to ABBR R851A. Each tube must be clearly labeled. The core will provide a cardboard tube box and will label the box with the correct project number.
5. The core will extract DNA from the supplied tissues if needed and resuspend the DNA in nuclease-free water. After genotyping is completed, the investigator can arrange with the core to have aliquots of the DNA for their own genotyping, if desired.
6. The core will perform standard PCR on the extracted DNA samples for the designed genotyping reactions.
7. Within two weeks of dropping off the specified number of samples, the core will provide the investigator with a genotyping report, including the total number of animals analyzed, total number of animals with genome editing detected, and total number of animals with desired genome editing event. If desired, the Investigator can arrange a consultation meeting to discuss the genotyping results. The core will provide advice as to which founders to breed from founder genotyping.
8. The investigator can decide whether they would like to continue with allele QA and have the core perform Sanger sequencing at an additional cost.  

What to Expect

1. The core will provide genotyping results from the previously designed schemes. If the PCR reactions fail to produce products with appropriate controls, new primers will be designed by the core and PCR will be repeated until genotyping results can be determined.
2. Indel and point mutations are often too small to detect by conventional PCR. Sanger sequencing and trace analysis will need to be performed to identify and characterize alleles produced. The core will recommend samples for Sanger sequencing, and will send out prepared samples with approval from the investigator. Sanger sequencing reactions will occur an additional charge per sample.
3. To verify any allele, Sanger sequencing of PCR products is necessary to verify the sequence at the target site(s). For N1 animals genotyped by the core, additional recommendations for samples for Sanger sequencing will be made. With approval from the investigator, new PCR amplicons will be generated and the core will send out prepared samples for Sanger sequencing. Sanger sequencing reactions will occur an additional charge per sample.
4. Founder animals are often mosaic. Thus, detection of desired genome editing events can be difficult at the founder generation. The core can make judgements as to which founders have targeted alleles, but the core cannot be completely certain without sequence confirmation.  

CRISPR/Cas projects initiated with the core will have PCR amplicons generated by the core for Sanger sequencing. Mice can be genotyped by the investigator and the core can perform subsequent Sanger sequencing. Alternatively, the core can perform the initial genotyping and investigators can add on the Sanger Sequencing service to sequence confirm desired alleles before starting to breed founder animals or to verify alleles in N1 animals. The core will analyze the sequencing traces and repeat the sequencing if the results are ambiguous. Tail clips, ear punches, or purified DNA samples can be submitted.

What Will Happen

If the core initially performed genotyping on the samples that will be Sanger sequenced, no additional action is needed by the investigator. The additional services will be added on to the existing genotyping request.

1. For stand-alone sequencing requests, the investigator will initiate a project in iLab. An account number must be provided at this time. The investigator will specify the number of animals they have for Sanger sequencing and fill out the sample submission table. Samples should be labeled using the following convention: Use the 4-digit iLab request number (i.e. GERM-DL-2578) followed by a dash and a three digit number from 001 to the number of N samples submitted. For example, 2578-001, 2578-002, etc. Samples submitted without this naming convention will have these labels assigned to the samples accordingly.
2. The core will have oligonucleotides for Sanger sequencing synthesized by an approved vendor. Primers that were used for previous genotyping by the core will be used if appropriate for Sanger sequencing.
3. The core will contact the investigator when it is ready to accept samples. Please do not submit samples until contacted by the core. Samples must be submitted in 1.5ml microfuge tubes to ABBR R851A. Each tube must be clearly labeled. The core will provide a cardboard tube box and will label the box with the correct project number.
4. The core will extract DNA from the supplied tissues if needed and resuspend the DNA in nuclease-free water. After Sanger sequencing is completed, the investigator can arrange with the core to have aliquots of the DNA for their own sequencing, if desired.
5. The core will perform standard PCR on the extracted DNA samples for the designed Sanger sequencing reactions.
6. The core will clean up the PCR reactions with the anticipated product size for the desired allele and send out samples for Sanger sequencing.
7. The core will analyze the Sanger sequencing results to confirm the sequence of the desired allele.
8. Within two weeks of dropping off the specified number of samples, the core will provide the investigator with a report, including the total number of animals analyzed, total number of animals sequenced, and total number of animals with desired allele. If desired, the investigator can arrange a consultation meeting to discuss the sequencing results.

What to Expect

1. The core will provide sequencing results from the previously designed genotyping schemes. If the PCR reactions fail to produce products with appropriate controls, new primers will be designed and PCR repeated before any sequencing is attempted.
2. If any sample fails to produce a quality trace by Sanger sequencing, repeat Sanger sequencing attempts will be submitted until the core can accurately assess the sequence for the sample at the specific locus.
3. Bioinformatics software is available to deconvolute heterozygous trace files containing indel and short HDR alleles in addition to wild-type sequence. This software will be used in place of traditional TA cloning to separate multiple PCR products for indel, epitope tag, point mutation, and conditional alleles. The core will not provide TA cloning services. 

CRISPR/Cas projects initiated with the core will be screened for potential off-target genome editing events in N1 animals using Sanger sequencing of PCR amplicons flanking predicted off-target sites. The core will design PCR primers to amplify a specified number of sites based on consultation with the investigator. Typically, the core recommends to screen 2-4 N1 mice at 5-10 off-target sites, however, there is no set number of mice or sites for each project. The core will analyze the sequencing traces and redesign the PCR scheme if the results are ambiguous.

What Will Happen

1. The investigator will initiate a project in iLab for Targeted Analysis of Off-Target Mutagenesis. The investigator will specify the number of animals they have for screening and the number of sites they wish to be screened. An account number must be provided at this time.
2. The investigator will collect tissue from the mice to be screened and arrange with the core to drop off the tissue to ABBR R851A. Alternatively, the investigator can supply the core with extracted DNA samples. The core requests an additional WT control sample for investigator extracted DNA. The control sample will not be charged for analysis.
3. The core will have oligonucleotides for PCR synthesized by an approved vendor.
4. If necessary, the core will extract DNA from the supplied tissues and resuspend the DNA in nuclease-free water. After screening is completed, the investigator can arrange with the core to have aliquots of the DNA for their own genotyping, if desired.
5. The core will perform standard PCR on the extracted DNA samples for the designed off-target sites.
6. The core will clean up the PCR reactions with the anticipated product size for the desired allele and send out samples for Sanger sequencing.
7. The core will analyze the Sanger sequencing results to confirm the absence heterozygous sequence. The presence of heterozygous sequence would indicate a genome editing event.
8. After analyzing the samples at all of the specified off-target sites, the core will provide the investigator with a report, including the genome locations of all off-target sites screened, the number of mismatches for each predicted site, the sequencing primers, and the results for each mouse analyzed by predicted site.

What to Expect

1. The core will provide off-target analysis results from predicted off-target sites primarily based on information available from the Wellcome Sanger Institute Genome Editing (WGE) Website (https://wge.stemcell.sanger.ac.uk/). This website predicts off-target sites with base mismatches to the target sequence and uses a canonical PAM (NGG). If the investigator wishes to screen off-target sites based on other prediction algorithms from guide RNA selection websites, they can provide the genomic coordinates for each predicted site to be screened.
2. If the PCR reactions fail to produce products with appropriate controls, new primers will be designed and PCR repeated before any sequencing is attempted.
3. Wild-type controls will also be sequenced to ensure that the Sanger sequencing results are the same as the predicted sequence from available genome browsers.
4. If any sample fails to produce a quality trace by Sanger sequencing, repeat Sanger sequencing attempts will be submitted until the core can accurately assess the sequence for the sample at the specific locus.