General Information about our program
Q: What type of services does the UC Davis Mouse Biology Program provide?
A: The Mouse Biology Program (MBP) develops and provides all necessary technologies and services for creating mutant mouse models, from concept to completion. Our services include, but are not limited to the following:
Q: Where can I find more information?
A: For additional information regarding the services we provide, please visit I want to, I need, and Help sections of the website. If you cannot find the answer to your question(s) there, please don’t hesitate to contact us at mbp@ucdavis.edu.
Q: Does the MBP provide its services to investigators outside of the University of California (UC) system?
A: Yes, the MBP routinely provides resources and services to UC investigators, as well as national and international scientists from approximately 480 universities, institutes or private industry. Please note at this time we cannot provide vector construction or electroporation services to ‘for profit’ institutions.
Q: Once I know which service I would like to pursue, how do I place an order?
A: Please download and complete the appropriate requisition form and return via email mbp@ucdavis.edu or fax to 530.757.3277. A list of available request forms is available here.
Q: Is there a discount associated with ordering multiple services?
A: The MBP is a non-profit, fee for service institution, which at this time is unable to provide a discount for multiple projects unless less there is actually less service work involved. All pricing is approved by our institution and based on actual labor and supplies required, without any mark-up. We do offer a 20% discounted rate to UC Davis Cancer Center members, on cancer related research, due to the Cancer Center Base Grant supplementation.`
Q: Who should I contact to get updates on my project?
A: To ensure a centralized and open communication channel between you and the various MBP laboratories, we have implemented a concierge level of assistance headed by our Project Concierge Sasha Stevens Wirth (mbp@ucdavis.edu).
Q: Do I need an IACUC protocol to make mutant mice?
A: If we make a transgenic or knockout mouse model for you, we will cover all the procedures and animals under our Animal Care and Use Protocol (#15723 Lloyd, expiration date available upon request). Once we have produced transgenic founders, chimeric or germline transmitted mice for you, we will transfer these mice to your protocol (and animal facility).
Transgenic Mouse Production
Q: What is involved in creating a transgenic mouse?
A: Transgenic mice are produced by pronuclear microinjection of fertilized eggs with a transgenic construct made by either the Mouse Biology Program (MBP) or individual investigator. Please click here for more information on transgenic constructs.
Q: What is the difference between a basic and guarantee pronuclear microinjection service?
A: For basic service, our Murine Targeted Genomics Laboratory (MTGL) will transfer 180 injected eggs, or get 40 live pups, or generate two founders, whichever comes first. The definition of “founder” is a mouse born from eggs that underwent pronuclear microinjection of a transgenic construct, and has at least one copy of the transgene.
For guarantee service, MTGL guarantees at least two founders. If no founders are produced, the investigator will not be billed. For this service, genotyping is to be performed by the Murine Genetic Analysis Laboratory (MGAL) at MBP.
Q: Is it possible to perform a co-injection procedure in order to produce multiple-transgenic mice?
A: Yes, the MTGL can perform co-injections to create double or triple transgenic mice. However, we cannot offer the guarantee service for this type of project, as the DNA concentration for each construct is only half of the concentration we use for standard procedures. For co-injections, mice with one transgene insertion will be considered founders.
Q: I am ready to send my transgene to MBP for pronuclear injection. What should I prepare to send?
A: Please see the Pronuclear Microinjection Request Form for more detailed information. In short, we look to receive restriction digested, gel purified, transgene fragment with little or no vector sequence. We will perform the final purification and dilution prior to injection.
Q: How long does it take to obtain founders from pronuclear microinjection?
A: The timeline for such a project varies depending on a number of things, including:
- Receipt of Pronuclear Injection Form, completed and signed by principal investigator
- Next available injection date
- Arrival of DNA at least one week before scheduled injection date
- The concentration and purity of the microinjected DNA supplied by the investigator
Q: What is the variation of copy number between individual founders generated from the microinjection? Can MBP test the founder lines for copy number?
A: Copy number varies greatly between founders, as each mutant is a distinct founder with potentially different expression and location of the transgene. Upon request, MGAL can perform copy number analysis on the founders, and we will report the results to the investigator.
Please see the Pronuclear Microinjection Request Form for additional information, or contact us at mbp@ucdavis.edu.
Targeted Mouse Production
Q: What are the steps involved in gene targeting?
A: A gene targeting construct (which contains a mutation in the gene of interest) is introduced into embryonic stem (ES) cells, by electroporation, in order to achieve a homologous recombination event which incorporates the null or mutant allele. After electroporation, the ES cells are grown under drug selection, picked, cultured, expanded, screened and confirmed for homologous recombination. The targeted clones are then assessed for euploidy by chromosome counts, and prepared for blastocyst microinjection, which allows for the creation of chimeric mice and further breeding for germ line transmission.
For more information about the gene targeting process and generation of targeted (KO, KI, cKO) mice please click here or contact us directly at mbp@ucdavis.edu.
Q: What is the timeline for generating targeted live mice, from concept to completion?
A: Currently, we estimate around 10-12 months for the production of chimeric mice. Germline transmission testing, to generate heterozygous animals, can add another 4-7 months to the timeline depending on the fertility and fecundity of the chimeras. We also offer an optional Flpe electroporation service to remove the positive selection marker in vitro, which adds approximately 3 months to the process. Although the timeline varies on a case by case basis, overall, we estimate between 14 and 22 months from vector construction through to germline transmission.
Q: Can the investigator supply the targeting vector for electroporation at MBP?
Yes, you can absolutely prepare the vector and send it to us for electroporation into ES cells. Please see the Electroporation Request Form for vector preparation instructions and additional details.
*Please note that in terms of targeting efficiency, it is usually best to target using isogenic DNA (i.e. C57BL/6 vector into C57BL/6 ES cells).
Q: What strain are the ES cells used for electroporation derived from?
We routinely use C57BL/6N ES cells (JM8.F6 and JM8A3.N1) or R1 ES Cells (129/SvJ) at our program and we have seen good germline transmission rates with both. For C57BL/6N, the rate is around 55% per clone which becomes increasingly efficient if more than one clone is injected. For example, if 2-3 clones are injected, the efficiency for germline transmission is around 90%. We generally inject 2-3 clones per project.
Q: How are ES cells screened for homologous recombination?
A: The ES cell clones are initially screened via a Loss-of-Allele (LOA) assay, which is a quantitative TaqMan® PCR that detects the loss of one region of the native target due to the correct recombination with the targeting vector. Once the initial LOA candidates are expanded from the 96 well plate, the clones are sent back to our genetics analysis laboratory where they undergo (1) selection cassette copy number analysis (ensuring there is only one copy of NEO and not a targeted as well as random integrant), (2) LoxP check (ensuring both LoxP sites remain intact with homologous recombination for conditional KO projects) and (3) Long Range PCR (PCR from vector/non genomic DNA to the outside of the long arm of homology).
Q: What is the difference between a basic and guarantee blastocyst microinjection service?
For our basic service, we promise minimum of 8 live pups born, or one male chimera equal or above 50%, whichever comes first. The guaranteed service guarantees at least one 50% male chimera is produced, or there will be no charge. This guarantee does not extend to germline transmission, however.
Q: Once chimeric mice are produced, which chimeras do you recommend breeding for germ line transmission?
A: The stem cell lines we inject to create the chimeric mice are XY stem cell lines. So it is considered a good sign when a majority of the high percentage chimeras are males. Female mice occasionally carry the targeted mutation in the germline, but only if the ES line has lost the Y chromosome, making it XO. Generally we suggest germ-line mating of two or three male chimeras between 70 and 90%.
Strain Cryoarchiving
Q: What is the most efficient and cost effective way of cryopreserving my mouse line?
A: In terms of storage, sperm cryopreservation is an inexpensive and efficient approach to cryopreserving your mouse line. For resuscitation services, frozen sperm can generally be recovered successfully by IVF followed by embryo transfer surgery. The success rates depend on the particular strain and sperm quality. In the more difficult cases, if the IVF fails, we have the capabilities to perform sperm resuscitation by intracytoplasmic sperm injection (ICSI).
Q: What are the advantages of embryo cryopreservation as compared to sperm cryo?
A: Embryo cryopreservation allows for the diploid genome to be preserved so that genotypically and phenotypically desired mice can be revived. Although the process is more time and cost demanding up front, the cost of strain resuscitation following embryo cryopreservation is much lower.
Q: Is genotyping carried out at MBP before cryopreservation?
No, we do not routinely genotype the imported mice before cryopreservation. It is the investigator’s responsibility to assess the correct strain and genetic nature of the line prior to submitting the strain cryopreservation request.
Q: Will the MBP be able to assist with distribution of the cryopreserved stock to my collaborators?
Yes, we are more than happy to coordinate shipment of the cryopreserved materials at the request of the investigator, or with permission from the investigator. Please note that the MBP does not function as a repository, and all genotyping and reference information would only be available from the requesting investigator. If you are interested in donating mice to a repository, please consider the MMRRC. A strain submission form is available at: http://www.mmrrc.org/submission/strain_submission_terms.html
Q: Where can I search for and purchase cryopreserved sperm or embryos for my gene of interest?
Frozen germplasm is available to purchase for a variety of transgenic, knockout, conditional knockout and gene trap mice. Please search the MMRRC and KOMP repository websites to determine if germplasm is available for your gene of interest.
