Transgenic Core Facility Services

  • Introduction
  • Animal Use
  • Fees and Forms
  • Sample Preparation
  • Services Offered
  • Custom Transgenic Mouse or Transgenic Rat Production
  • Bacterial Artificial Chromosome Engineering
  • Gene Targeting Service
  • ES Cell-Mouse Chimera Production
  • De Novo ES Cell Line Derivation
  • Personnel Training
  • Plasmids, ES Cells, and Culture Reagents
  • Material Transfer Agreements
  • Centralized Support
  • Conversion of Mouse Lines to Specific Pathogen Free Status
  • Embryo Cryopreservation/Recovery
  • Speed Cryo
  • In Vitro Fertilization
  • Sperm Cryo


  • Introduction
    The University of Michigan Transgenic Animal Model Core was established in 1989 in response to the need for transgenic technology by University Investigators. The mission of the Transgenic Core is to provide access to transgenic animal technology in an efficient, effective manner. This includes 1) the production of transgenic mice, 2) the production of transgenic rats and zinc finger nuclease gene knockout and knockin rats, 3) the production of gene-targeted mouse embryonic stem (ES) cells for mouse knockout models and in vitro studies, 4) the production of ES cell-mouse chimeras for mouse knockout models, 5) assisted reproduction technology for mice and rats, 6) de novo derivation of mouse ES cells from custom mice, and 7) hands-on training in mouse ES cell manipulation, pronuclear microinjection, and blastocyst injection. Researchers can use Transgenic Core laboratory space and resources side by side with staff members. Consultation in all phases of transgenic and gene targeting research from experimental design to mouse breeding is freely available. Meet the Staff.

    The Transgenic Core collaborates with the BAC Recombineering Core to prepare genetically modified bacterial artificial chromosomes (BACs). The mission of the BAC Core is to provide access to a complex technology to investigators at the so that they can use their resources to conduct research instead of developing tools for research. BACs are genetically modified by homologous recombination (BAC recombineering) to prepare DNA mutations, insertions and deletions. BACs can be used as transgenes to express reporter molecules (GFP), Cre recombinase, and mutant alleles in cell culture and mice. The BAC Core also prepares gene targeting vectors for the modification of chromosomes in ES cells.

    The Transgenic mouse core guarantees that at least three transgenic founder mice will be produced for each DNA construct. The overall average is ten founders. Since 1989 over 14,000 mouse transgenic founders were produced from more than 1,400 plasmid and BAC transgenes. Nearly 1000 transgenic rat founders have been produced, including BAC transgenic rat strains. Over 200 new mutant mouse models were produced by the electroporation of mouse ES cells with targeting vectors and another 60-odd strains were produced from ES cell clones obtained from the international mouse knock out project (KOMP). Some of these mouse and rat models have been published.

    University Committee on the Care and Use of Animals
    Prior to the production of genetically altered animals, investigators must have approval to use mice in their research from the University Committee on Use and Care of Animals.

    Fees
    This facility exists to serve the needs of University of Michigan researchers. A list of fees is availalbe. Investigators are invited to contact Thom Saunders with any questions. The genetically altered animals provided by this facility can only be used for research purposes. Access to cell lines and plasmids is contingent upon approved material transfer agreements. Request  forms are available for download.

    Sample Preparation
    For the production of conventional transgenic mice, investigators are responsible for providing a restriction digest containing 50 ug of the transgene insert. The Transgenic Core will purify DNA for microinjection as described. A minigel photo should accompany the digest, the DNA fragment to be purified should be clearly marked. Several things should be considered in designing a transgenic research project (see Transgenic Project Outline). Prokaryotic vector sequences interfere with the expression of some transgenes, thus unique restriction sites at the 5' and 3' ends of the construct should be available for vector removal. The transgene should contain unique markers so that its presence can be easily detected in DNA samples and its expression can be assayed and distinguished from endogenous gene expression.

    Certain transgenes may result in a very low yields of transgenic founders due to the intrinsic nature of the transgene. For example, certain genes will be deleterious or incompatible with proper growth and development of the embryo. Special arrangements should be made with the Core if the transgene is suspected to cause lethality. The expression of a transgene requires that the appropriate transcriptional control elements be included in the DNA construct. Expression is often influenced by the chromosomal site in which the transgene DNA is integrated. Preliminary studies in cell cultures are recommended to verify the integrity of the construct and the function of the promoter/enhancer. However, it is not always possible to predict in advance whether the transgene will be expressed in vivo. For these reasons, the Core cannot guarantee that transgenic founders will express the transgene.

    For the production of gene targeted mice with mutations induced by homologous recombination in ES cells, investigators are responsible for producing ES cell clones with targeted genetic mutations. The first step in gene targeting is to obtain a detailed restriction map of the genetic locus and knowledge of exon/intron boundaries. (see Gene Targeting Outline) Higher yields of targeted ES cell clones are obtained from isoegenic DNA, thus a clone(s) from a genomic library that exactly matches the ES cell line to be electroporated is strongly advised. Restriction mapping data is used to identify 5' and 3' arms for targeting vector construction and to establish a screening strategy for the identification of ES clones that have undergone homologous recombination. This requires the characterization of probes for Southern blot analysis that lie outside of targeting vector sequences. After the targeting vector has been cloned it is introduced into ES cells. Fastidious cell culture technique and specialized reagents are required to maintain ES cells in a pluripotent state. Differentiated ES cells will not produce chimeras with the ability to transmit targeted mutations through germ cells. Investigators are invited to contact Elizabeth Hughes for training in ES cell culture and to obtain reagents certified for ES cell culture. Due to the intrinsic variability of individual ES cell clones we can not guarantee that germline chimeras will be produced from any particular ES cell clone. The Transgenic Facilty tests and validates ES cell culture reagents to maximize the successful outcome of gene targeting projects. 


    Services Offered: View Detailed Service List

  • Custom Transgenic Mouse Models: 3 transgenic founders are guaranteed
  • Custom Transgenic RAT models: 3 transgenic founders are guaranteed
  • Bacterial Artificial Chromosome Recombinering Core
    Gene Targeting Service: We work with you to "knockout" a gene in a mouse.
    Personnel Training
  • Plasmids and certified ES cell culture reagents
  • Centralized Support for transgenic animal production
  • Conversion of mouse lines to specific pathogen free status
  • Embryo cryopreservation and recovery



  • Service Descriptions
    1. Custom Transgenic Mouse or Transgenic Rat Production: The Core will carry out all the steps necessary to produce transgenic mice or rats  from cloned DNA provided by the investigator.The Transgenic Facility will purify
    DNA for microinjection from a restiction digest provided by the investigator. A minimum of three transgenic founder animals will be produced for each DNA construct. Orders must be canceled two weeks in advance in order to avoid a cancellation fee. The investigator is responsible for providing the following materials:
    1. a restriction digest that contains 50 ug of the transgene in a restriction digest.
    2. a minigel photo of the digest that shows the digest is correct and which shows which DNA fragment is the transgene.
    3. a PCR assay that demonstrates that the transgene DNA can be detected at the single copy level when mixed with in mouse or rat tail tip DNA.
    4. a PCR assay that demonstrates that a single copy gene can be amplified from mouse tail DNA or rat tail DNA.
    5. a complete billing form, contact Thom Saunders  for a blank form or  download the form.
    A recommended protocol for microinjection DNA purification is available. If requested, the Core will assist labs in establishing methods of transgene detection. The Core will purchase the required mice and pay for their housing, including vasectomized males and females for foster mothers. The mice are housed under specific pathogen free conditions. Fertilized (C57BL/6 X SJL)F2 mouse eggs will be collected, microinjected, and transferred to pseudopregnant recipients. When the pups are two weeks old the Core will ear tag the mice and deliver tail biopsies to the investigator for transgene detection. One week after delivering the biopsies, the Core will wean the mice and transfer them to the investigator. Upon request the Core will demonstrate the animal identification and tissue sampling techniques to the investigator for use in maintaining the line. Requests for production in other mouse strains will be accommodated with advance approval. In such cases additional costs for the purchase and housing of egg donors and studs from may be necessary. Contact Thom Saunders with any questions.

    2. Bacterial Artificial Chromosome (BAC) Recombineering Services: The Transgenic Facility includes a BAC Recombineering Core. BACs containing genes of interest are obtained from BAC library resources. Homologous recombination of DNA fragments is used to modify BAC sequences so as to produce gene knockins, gene knockouts, piont mutations, or to generate gene targeting vectors. Specialized bacterial strains and plasmid reagent have been obtained from the NCI for this purpose. Additional plasmids useful for recombineering are under development in the BAC Core. Modified or native BACs can be used as transgenes in the generation of BAC transgenic mice or BAC transgenic rats. BACs of any species for which the complete sequence is known can be modifed. Contact Michael Zeidler or  Thom Saunders with any questions.

    3. Gene Targeting Service: This is a collaborative service combining the knowledge of the investigator with the specialized skills of the Transgenic Core. This service produces embryonic stem cells with mutations induced by homologous recombination with a targeting vector provided by the investigators. The stem cells can be used to generate germline ES cell-mouse chimeras and mice with novel mutations. Some may wish to conduct in vitro differentiation studies. It allows investigators to focus on the molecular biology pf the gene in question while the Core focuses on generating pluripotent embryonic stem cells with the targeted mutation. This collaborative approach emphasizes the strengths of each partner and obviates the need for laboratory personnel to master the fastidious technique that is necessary to culture totipotent mouse embryonic stem cells. Contact Thom Saunders with any questions.

    The investigator performs the following tasks: Restriction map the endogenous gene. Establish a robust, reliable, reproducible screen that discriminates between wild type and targeted alleles. Provide experimental data demonstrating that the Southern blot probes confirm the predicted genomic structure of targeted ES cell clones. Clone the gene targeting vector or make arrangements with the BAC Recombineering Core to prepare the targeting vector - contact Contact Thom Saunders for BAC Core information. Purify gene targeting vector DNA for electroporation according to the protocol supplied by the Transgenic Core. Analyze genomic DNA from as many as 480 ES cell clones for homologous recombination with the targeting vector. Timely analysis is important because of the limited life span of ES cells cryopreserved at -80 degrees C. Review the screening data with the Transgenic Core prior to expansion of cryopreserved clones.

    Transgenic Core staff will provide/perform the following information/procedures: Prepare an experimental time line for planning purposes. Electroporate the targeting vector into ES cells. Pick 480 electroporated ES cell clones (five 96-well plates). Each plate of the five 96-well plates will be split into three. Two plates will be cryopreserved in independent -80 degree C freezers. One plate will be grown and split into two replicates for DNA preparation. Prepare DNA from replicate 96-well plates (ten plates total, two replicates of each of the five plates of clones). Deliver the DNA plates to the investigator for screening.

    Once ES cell clones with targeted mutations are identified, Core staff will continuously expand ES cell clones until 5 vials of 5 X 106 cells/ml can be produced for storage in liquid nitrogen. Each clone will be tested for mycoplasma and morphology evaluated. Clones will be chromosome counted to identify euploid clones and ES cell pellets will be provided to the investigator DNA extraction and  verification of targeting. Clones that meet the criteria for microinjection (euploid, good morphology, clear of infection, correctly targeted) can be scheduled for microinjection into blastocysts for chimera production.

    4. ES Cell-Mouse Chimera Production: The Core will inject C57BL/6 blastocysts with embryonic stem cells (129 mouse derived ES cell clones) or albino C57BL/6 ES blastocysts (C57BL/6 ES cell clones). A minimum of 60 blastocysts will be injected with each ES cell clone. Orders must be canceled two weeks in advance in order to avoid a cancellation fee. The Core will purchase the required mice and pay for their housing, including vasectomized males and females for foster mothers. The mice are housed under specific pathogen free conditions. Blastocysts will be collected, injected with 10 to 16 ES cells, and transferred to pseudopregnant recipients. When the chimeric pups are three weeks old the Core will transfer the mice to the investigator. If desired the investigator can make arrangements with the Transenic Core for germline breeding of ES cell-mouse chimeras.. The generation of chimeric mice is highly dependent on the condition of the ES cells. Thus, we can not guarantee germline chimeras will be produced from each ES cell clone. Investigators interested in  culturing their own ES cells for blastocyst microinjedion are encouraged to contact Elizabeth Hughes regarding ES cell lines and their culture requirements.

    5. DeNovo ES Cell Line Derivation. The Transgenic Core will prepare new mouse ES cell lines from blastoycsts or mice provided by investigators. Standard methods employing serum containing medium and MEK1 inhibitor are used. The success rate of this procedure is very high (100% success to date) as long as blastocysts can be obtained from the mouse strain in question. Mouse ES cells provide a endless supply of cells for in vitro studies because ES cells do not undergo senesence and cease division as do other cell types (e.g. fibroblasts). In addition is possible to differentiate ES cells into different lineages to query gene function in cell culture systems instead of or in parallel to in vivo studies. The procedures requires intensive cell culture over an extended period of time by Transgenic Core personnel.Advance notice should be given, preferably when the blastocyst-donors-to-be are born. The ideal age for of in-house blastocyst donors for superovulation response is 24-28 days. A request form can be downloaded here. Contact Thom Saunders  well in advance to schedule this procedure. Training in ES cell culture is available for those who are interested. See Personnel Training.

    6. Personnel Training: Training in the production of genetically altered mice and ES cell culture are offered:
        Pronuclear Microinjection Training: Individuals will be trained in two stages: first, embryo collection and reimplantation, and second, microinjection of fertilized eggs with transgene DNA.  BAC Transegnic inject training is available upon request. 40 hours of training is provided. Transgenic Rat training is available in addtion to transgenic mouse production training. 40 hours (1 week) of training is provided.  It is not unusual for investigators to generate transgenic founders during the training week.See the sample Mouse Syllabus and/or the sample Rat Syllabus.
        Blastocyst Microinjection Training: Individuals will be trained in two stages: first, embryo collection and reimplantation, and second, microinjection of blastoycsts with ES cells. 40 hours (1 week) of training is provided. See the sample Syllabus.
        Mouse ES Cell Training Class: Those with prior experience in handling mice will require less time. Individuals will be trained in all aspects of ES cell culture and manipulation. They will learn every technique necessary to successfully produce ES cell clones with targeted mutations induced by homologous recombination. Investigators are expected to present relevant papers and discuss papers that provide the experimental basis for procedures learned in the laboratory setion of the class. 80 hours (two weeks) of training is provided.  See the sample Syllabus.
        Contact Thom Saunders to schedule training.

    7. Plasmids, ES Cells, and Culture Reagents: Plasmids and ES cell reagents are maintained by the Core. The following plasmids are available: pnlacf, which contains beta galactosidase as a reporter gene; pPNT, a plasmid for the construction of gene targeting vectors; the pflox vector designed for the preparation of mouse models with tissue specific gene inactivation. A CMV-Cre plasmid (pBS185) is commercially available from Addgene. The Core has numerous germline competent mouse ES cell lines available from both 129 mouse strains and C57BL/6 mouse strains. Others can be obtained through KOMP.  In addition, the Core tests fetal bovine serum (FBS) for lots which will support ES cell proliferation with minimal differentiation and stores a large quantity for resale at cost. Feeder cells for ES cell culture are prepared from neor transgenic mice to optimize culture conditions during positive selection. Please contact Thom Saunders with any questions on how to initiate material transfer agreements for these reagents.

    8. Centralized Support: This consists of access to microinjection and ES cell culture workstations. Microinjection support includes quality tested media for egg and blastocyst culture, quality tested hormone stocks, equipment maintenance, and all of the miscellaneous plastic and glass supplies. Users will be responsible for maintaining their own animals for embryo donors, male studs, vasectomized males, and pseudopregnant females. Plasticware for tissue culture of ES cells is provided at cost. Users will be responsible for providing their own media.Pluripotent ES cells, tested FBS, and feeder cells are available from the Core at nominal cost (see 4. above). Please contact Thom Saunders to schedule equipment use.

    9. Conversion of Mouse Lines to Specific Pathogen Free Status: The Core will obtain fertilized eggs from pathogen infected mice and transfer them into clean, specific pathogen free (SPF) recipients as described (Van Keuren and Saunders, 2004). The investigator is responsible for providing mice of the strain in question. The Core will transfer washed, fertilized eggs to SPF pseudopregnant females. The mice will be tested to verify the SPF status of the offspring. This method eliminates viral (e.g. murine hepatitis virus, Sendai virus, parvovirus), bacterial (e.g. mycoplasma pulmonis),  ectoparasite (mites), and endoparasist (pinworm) infections. We have converted many different mouse strains to SPF status by this procedure. Our success rate is 100%, in every case the foster mothers and offspring have been free of infection. Please contact Thom Saunders if you need to convert mice to pathogen free status.

    Van Keuren ML, Saunders TL. 2004.Rederivation of Transgenic and Gene-Targeted Mice by Embryo Transfer. Transgenic Res. 13:363-371.

    10. Embryo Cryopreservation/Recovery: The purpose of embryo freezing is to protect against the loss of valuable, unique mouse stocks through breeding failure or disease, and to eliminate the cost of maintaining mouse lines not actively in use. Investigators will provide the Transgenic Core with stud males and egg donors from the line or stock which they desire to preserve. Typically 15 stud males are mated weekly with egg donors per cryopreservation session to produce embryos for cryopreservation. On average, it takes 4 embryo collection sessions to freeze down enough embryos to guarantee recovery. If you can provide us with homozygous males then fewer sessions will be needed. However, it may take more sessions if the mouse strain has a low superovulation rate, the stud males have low fertility, or males are too old. Some strains can not be successfully cryopreserved.  The investigator is responsible for providing all stud males, embryo donors, and per diem costs for these animals. The Core will collect and freeze fertilized mouse eggs at the eight cell stage. A test batch of embryos will be thawed and transferred to pseudopregnant recipients from each cryopreservation session. Frozen embryos will be maintained in liquid nitrogen and an annual storage fee will be assessed. Please contact  Galina Gavrilina (or 763-6209) if you wish to cryopreserve or recover stocks.

    How much does it cost?
    Our recharge is based on a set fee per cryopreservation session. A cryopreservation session means that we collect embryos from 15 donors, freeze down 8-cell embryos, test thaw some of the embryos, culture the thawed 8-cell embryos to blastocyst, transfer the blastocysts to recipients, and count then number of fetuses or pups that result. We may ask you test DNA from the pups to verify their genetic composition.

    How many cryopreservation sessions does it take to freeze a line?
    Assuming that you have 15 singly housed hemizygous transgenic mice and you are using C57BL/6 egg donors it will take about 3 or 4 sessions to store 600 cryopreserved embryos (300 transgenic embryos) in liquid nitrogen. If the stud males are homozygous for the gene then fewer sessions will be needed since we can stop after we freeze down 300 embryos. If the males have poor fertility and we obtain fewer than 100 embryos per cryopreservation session, it will be more cost-effective to replace the males than to do 6 or more sessions to generate the needed embryos to cryopreserve your line.

    Why do you need to freeze so many embryos?
    This standard is widely adopted by commercial providers. Our goal is to freeze down the embryos and guarantee that we can bring the mice back from cryopreservation in the future. This lets you euthanize all of the mice on the shelf in your mouse room while being assured of access to the animals for your future research.

    What if we can't generate 300 transgenic/knockout embryos to cryopreserve my mouse strain?
    Our goal is to bank 300 embryos with the desired genotype.  This allows for long-term storage of your embryos in multiple sites for the highest level of security and recovery. If we are unable to generate 300 embryos because of poor reproductive performance we may still be able to recover your mice from cryopreserved embryos once or twice, depending on the number of embryos frozen and their performance on thawing.

    Who covers the animal purchase and per diem costs?
    You do. Galina Gavrilina (763-6209) will sit down with the person who is managing your mouse colony and work out a schedule for mouse deliveries. You need to provide 15 fertile stud males and order in a series of egg donors for superovulation and mating to the studs. Galina will collect the 8-cell embryos after mating, freeze them down, and test thaw them.

    Can I go ahead and do a freeze if I have only 6 to 12 males?
    Based on our experience, this is costly, inefficient, and very time consuming. With so few males available, fewer eggs for cryopreservation will be produced each session. This means that a greater proportion of the eggs will be lost to test thaws. Simply put, we need you to provide 15 stud males for us to go forward.

    What if I have an FVB/N transgenic line that I want to freeze down?
    We can freeze down FVB/N lines. However, it may require more than 3 or 4 sessions because FVB/N female mice produce fewer eggs than C57BL/6 eggs in response to the superovulation treatment.

    11. Speed Cryo
    Speed Cryo is a method to cryopreserve mouse strains. Cryopreservation safeguards mouse strains against breeding failures, pathogens, and genetic contamination. Speed Cryo relies on in vitro fertilization (IVF) to generate 2-cell embryos for cryopreservation. Our goal is to bank 300 embryos with the
    desired genotype.  Once a strain is banked, un-needed live stocks of mouse can be eliminated.

    Compared to the standard approach of cryopreserving 8-cell embryos, Speed Cryo requires fewer males to generate embryos for cryopreservation. A single session with 2-4  homozygous males and C57BL/6 egg donors can generate 300 embryos for cryopreservation. If heterozygous males are used, it is likely that two or more Speed Cryo sessions will be needed to produce 300 embryos for cryopreservation. The advantage of Speed Cryo is that many embryos can be generated from a single IVF procedure. The disadvantage of the Speed Cryo approach is that it depends on good IVF yields. The efficiency of mouse IVF varies depending on the strain background and the fertility of the individual males used as sperm donors (Byers, et al., 2006, Vergara et al., 1997). Thus, it is possible that IVF with transgenic or knockout sperm will produce few eggs for cryopreservation even though a control IVF with hybrid mouse sperm gives good results. In these cases, it is advisable to repeat the Speed Cryo procedure to control for variability in the transgenic or knockout males. If too few eggs are generated  then  the remaining option is generate and freeze down 8-cell embryos.

    Byers SL, Payson SJ, Taft RA. 2006. Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs).Theriogenology. 65:1716-26.

    Vergara GJ, Irwin MH, Moffatt RJ, Pinkert CA. 1997. In vitro fertilization in mice: Strain differences in response to superovulation protocols and effect of cumulus cell removal. Theriogenology 47:1245-1252.

    The Transgenic Core will superovulate 25 C57BL/6 egg donors (other strains can be substituted) perform IVF with sperm from the males you donate to us and 20 C57BL/6 egg donors. We will do a positive IVF control with sperm from (C57BL/6 X DBA/2)F1 male and 5 C57BL/6 egg donors.  After overnight culture,  2-cell embryos will be frozen stored in liquid nitrogen. When sufficient numbers of embryos are present, they will be divided between two liquid nitrogen containers in two different buildings. We will perform a test thaw on each batch of cryopreserved embryos. Thawed embryos will be scored for survival and transferred to pseudopregnant females.  The females will be scored for pregnancy and the tissue from the pups will be provided to the investigator. We expect the investigator to genotype the pups and determine whether the pups have the desired  genotype.  Contact Galina Gavrilina (or 763-6209) to schedule Speed Cryo procedures. Download Speed Cryo Request Form.

    Speed Cryo Cost
    We expect investigators to provide sperm donors and to pay for the purchase of egg donors.  In addition to the service fee you will be recharged for the purchase costs of the egg donors. The complete fee covers  the IVF procedure, cryopreservation of 2-cells eggs, storage in monitored liquid nitrogen vessels, and a test thaw and transfer of frozen eggs to verify viability.

    12. In Vitro Fertilization
    Mouse In Vitro Fertilization (IVF) can be used to rapidly expand mouse lines from a few males that carry the desired genotype or  to  maintain strains  with poor breeding efficiency. The Transgenic Core will superovulate 25 C57BL/6 egg donors per session  (other strains can be substituted). We will  perform IVF with sperm from your males and 20 C57BL/6 egg donors. We will perform a positive IVF control with sperm from (C57BL/6 X DBA/2)F1 males and 5 C57BL/6 egg donors. After overnight culture,  2-cell embryos will be transferred to pseudopregnant females.  All weaned pups will be transferred to the investigator.  We expect the investigator to genotype the pups and determine which pups have the desired genotype(s). 

    IVF results vary according to genetic background and the quality of individual males used for IVF (Byers et al., 2006, Vergara et al., 1997). Thus we can not offer a guarantee that any given IVF procedure will produce large number of pups. The standard recharge for each IVF session is the cost of purchasing the egg donors and a fee to recover our costs. If both the experimental and the control IVF procedures fail then we will not recharge your account. Contact Galina Gavrilina (or 763-6209) to schedule IVF procedures. Download IVF Request Form.

    Byers SL, Payson SJ, Taft RA. 2006. Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs).Theriogenology. 65:1716-26.

    Vergara GJ, Irwin MH, Moffatt RJ, Pinkert CA. 1997. In vitro fertilization in mice: Strain differences in response to superovulation protocols and effect of cumulus cell removal. Theriogenology 47:1245-1252.

    13. Sperm Cryopreservation
    Mouse sperm cryopreservation is offered as a routine service. Request forms are available. We will collect sperm from your male(s) and freeze down 10 aliquots per male and store them in liquid nitrogen. We recommend that you order an IVF service to test the sperm aliquots for their ability to generate viable pups with the desired genotype. Recovery of live mice from frozen mouse sperm is strain dependent (Thornton et al., 1999).

    Thornton CE, Brown SD, Glenister PH. 1999. Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses. Mamm Genome. 10:987-992.


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