Transgenic Animal Model
Custom Transgenic Mouse or Transgenic Rat
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 restriction 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:
- a restriction digest that contains 50 ug of the transgene in a restriction digest.
- aminigel photo of the digest that shows the digest is correct and which shows which DNA fragment is the transgene.
- aPCR 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.
- a PCR assay that demonstrates that a single copy gene can be amplified from mouse tail DNA or rat tail DNA.
- a complete billing 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.
Bacterial Artificial Chromosome (BAC) Recombineering
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 modified.
Gene Targeting Service
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.
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 - contactThom 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.
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 Transgenic 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 cannot guarantee germline chimeras will be produced from each ES cell clone. Investigators interested in culturing their own ES cells for blastocyst microinjection are encouraged to contact Elizabeth Hughes regarding ES cell lines and their culture requirements.
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. Contact Core staff well in advance to schedule this procedure. Training in ES cell culture is available for those who are interested.
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.
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.
Conversion of Mouse Lines to Specific Pathogen
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.
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 cannot 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.
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 Cryois 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.
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.
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.
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 cannot 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.
Mouse sperm cryopreservation is offered as a routine service. 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).
- Space in the ES cell lab can be provided to labs that want to produce targeted ES cells.
- Microinjection workstations are available for labs that want to do their own injections.
- Adherent cell microinjection workstation equipped with EppendorfInjectman System for Microinjection of Adherent Cells. This consists of a Model 5179 Micromanipulator and a FemtoJet 5247 Microinjector.
- Conversion of Mice to Specific Pathogen Free Status by embryo transfer.
- Mouse strain cryopreservation.
- Recovery of Cryopreserved Mouse Embryos.
- Consulting on transgene design, targeting vector design, and experimental plan.
- Specialized research projects that take advantage of our equipment and expertise such as intraplacental microinjection.
- We guarantee the production of transgenic mice, but cannot guarantee that any given transgene will be expressed in transgenic mice.
- We guarantee a minimum number of blastocysts will be injected with ES cells, but cannot guarantee that any given ES cell clone will form germline mouse chimeras.
- We guarantee that the gene targeting reagents are germline competent.
- The Genetically engineered animals provided by this facility can only be used for research purposes.
- Access to cell lines and plasmids is only provided to investigators who have complete, approved material transfer agreements.
- Investigators must have approval to use animals in research and teaching from the University Committee on the Care and Use of Animals.