Injection of mouse embryonic stem cells into blastocysts/Service Request form plus documentation.

Policy:

Blastocyst Injection

ES cells provided to the facility will be injected after visual inspection of their morphology.  Cells that are in poor condition will not be injected, and investigators may be charged an additional fee for blastocysts collected. To avoid unnecessary animal use, we request a week's notice for cancellation of an injection date. For cells that were not produced in the GEMF, we will guarantee injection of 50 blastocysts, but cannot guarantee chimeras.

Isolation and cryopreservation of mouse embryos/Service Request form.

Policy:

Cryopreservation

While cryopreservation is a valuable tool for conserving mouse resources, certain mouse strains are not amenable to the rigors of the technique. For that reason, it is imperative to know the genetic background of the strain to be cryopreserved. The GEM Facility will thaw one straw of frozen embryos and culture overnight to the blastocyst stage to insure that embryo freezing is feasible for that particular mouse line. For sperm cryopreservation, the investigator will be given a schedule for pre-mating the males to encourage new sperm production.

Implantation of fresh or previously frozen embryos into pseudopregnant females/Service Request form.

Expansion of ES cells generated outside of MDACC and cytogenetic analysis/Service Request form.

ES cell generation will include the isolation of the inner cell mass and subsequent growth of ES cells from blastocysts obtained from five to ten superovulated females.

Gene targeting to obtain a knock-in mutation using CRISPR/Cas9, TARGATT^@, ZFNs

• Injection of sgRNAs, Cas9 mRNA or protein and a DNA construct into pronuclear stage mouse embryos to generate founder animals. Embryo donors may be wild-type or obtained from an investigator’s own mouse line for additional mutation.
• Injection of ZFNs and a DNA construct into pronuclear stage mouse embryos to generate founder animals. Embryo donors may be wild-type or obtained from an investigator’s own mouse line for additional mutation.
• Injection of PhiC31 integrase and the att-b flanked DNA construct into pronuclear stage TARGATT^@ mouse embryos to generate founder animals.

Electroporation of investigator-provided construct into mouse embryonic stem cells/Service Request form plus documentation.

Policies:

Recombinant DNA Experiments

Because pronuclear injections and ES cell electroporations utilize unique DNA sequences that recombine into the mouse genome, the principal investigator needs to provide the GEM Facility with an approval number from the Institutional Biosafety Committee (IBC) prior to beginning these experiments.

Gene-Targeting in ES Cells

The investigator is responsible for demonstrating a reliable scheme for identifying targeted ES cell clones by Southern analysis with 5' and 3' probes by providing results from pilot experiments for this purpose. PCR analysis may be suitable for analysis if appropriate control experiments have been done. (Call Jan Parker-Thornburg for further information on certifying PCR analysis.) A convincing strategy for identifying mutant alleles must be demonstrated prior to the initiation of a gene-targeting experiment. 

Fertilization of mouse oocytes using frozen or fresh mouse sperm in vitro. The investigator should advise the GEMF of the mouse requirements from the IVF, whether only a small number of mice are required, or a large cohort of mice is required.

As it can often be difficult for investigators to produce 15-20 fertile males for embryo cryopreservation, we also offer the option of an IVF-Cryopreservation procedure. For this procedure, we will require one or two optimally fertile males. The males will be sacrificed to obtain sperm that is then mixed with oocytes in vitro. Two-cell embryos resulting will be vitrified and stored for future use. While this procedure is much easier on the investigator, there will often be long waiting times for this service due to the amount of technical time required. As in the embryo cryopreservation procedure, we will archive 200 embryos from a heterozygous x wild-type mating and 100 embryos from a homozygous x wild-type mating. All procedures will be tested by thawing a small aliquot of embryos and determining survival.

Injection of investigator-provided DNA construct into pronuclear stage mouse embryos/Service Request form plus documentation.

Policies:

Recombinant DNA Experiments

Because pronuclear injections and ES cell electroporations utilize unique DNA sequences that recombine into the mouse genome, the principal investigator needs to provide the GEM Facility with an approval number from the Institutional Biosafety Committee (IBC) prior to beginning these experiments.

Pronuclear Injection

Our facility has produced thousands of founder lines by pronuclear injection. While DNA can generally be shown to incorporate into the genome, the gene may or may not exhibit expression. It is not always possible to predict whether the transgene will be expressed and the effects of its expression in the mouse. For this reason, the GEM Facility does not guarantee transgene expression. The investigator is responsible for demonstrating a reliable scheme for identifying detection of the transgene at a single copy level by providing results from pilot experiments for this purpose. The GEMF will provide protocols for single-copy detection upon request.

Generation, isolation and implantation of mouse embryos into pseudopregnant females/Service Request form.

Isolation and cryopreservation of mouse sperm.  The GEMF will cryopreserve sperm from at least 2 males of prime breeding age.  Investigators will be requested to set up a timed mating just prior to the procedure (on a timeline designated by the technician) in order to insure the production of fresh sperm. All sperm cryo procedures are validated by subsequent IVF and culture to 2-cell.

Policy:

The background strain of the mice for freezing plays an important role in the success of the procedure. We may not be able to perform some procedures in certain strains of mice. Therefore, this information is critical for us to determine the potential success of your project.

Electroporation of investigator-provided construct with 10-15 colonies picked for analysis/Service Request form.

Pricing is available upon request, after consultation with the investigator to determine the requirements of the specific project.

Injection of mouse embryonic stem cells into blastocysts/Service Request form plus documentation.

Policy:

Blastocyst Injection

ES cells provided to the facility will be injected after visual inspection of their morphology.  Cells that are in poor condition will not be injected, and investigators may be charged an additional fee for blastocysts collected. To avoid unnecessary animal use, we request a week's notice for cancellation of an injection date. For cells that were not produced in the GEMF, we will guarantee injection of 50 blastocysts, but cannot guarantee chimeras.

Isolation and cryopreservation of mouse embryos/Service Request form.

Policy:

Cryopreservation

While cryopreservation is a valuable tool for conserving mouse resources, certain mouse strains are not amenable to the rigors of the technique. For that reason, it is imperative to know the genetic background of the strain to be cryopreserved. The GEM Facility will thaw one straw of frozen embryos and culture overnight to the blastocyst stage to insure that embryo freezing is feasible for that particular mouse line. For sperm cryopreservation, the investigator will be given a schedule for pre-mating the males to encourage new sperm production.

Implantation of fresh or previously frozen embryos into pseudopregnant females/Service Request form.

Expansion of ES cells generated outside of MDACC and cytogenetic analysis/Service Request form.

ES cell generation will include the isolation of the inner cell mass and subsequent growth of ES cells from blastocysts obtained from five to ten superovulated females.

Gene targeting to obtain a knock-in mutation using CRISPR/Cas9, TARGATT^@, ZFNs

• Injection of sgRNAs, Cas9 mRNA or protein and a DNA construct into pronuclear stage mouse embryos to generate founder animals. Embryo donors may be wild-type or obtained from an investigator’s own mouse line for additional mutation.
• Injection of ZFNs and a DNA construct into pronuclear stage mouse embryos to generate founder animals. Embryo donors may be wild-type or obtained from an investigator’s own mouse line for additional mutation.
• Injection of PhiC31 integrase and the att-b flanked DNA construct into pronuclear stage TARGATT^@ mouse embryos to generate founder animals.

Electroporation of investigator-provided construct into mouse embryonic stem cells/Service Request form plus documentation.

Policies:

Recombinant DNA Experiments

Because pronuclear injections and ES cell electroporations utilize unique DNA sequences that recombine into the mouse genome, the principal investigator needs to provide the GEM Facility with an approval number from the Institutional Biosafety Committee (IBC) prior to beginning these experiments.

Gene-Targeting in ES Cells

The investigator is responsible for demonstrating a reliable scheme for identifying targeted ES cell clones by Southern analysis with 5' and 3' probes by providing results from pilot experiments for this purpose. PCR analysis may be suitable for analysis if appropriate control experiments have been done. (Call Jan Parker-Thornburg for further information on certifying PCR analysis.) A convincing strategy for identifying mutant alleles must be demonstrated prior to the initiation of a gene-targeting experiment. 

Fertilization of mouse oocytes using frozen or fresh mouse sperm in vitro. The investigator should advise the GEMF of the mouse requirements from the IVF, whether only a small number of mice are required, or a large cohort of mice is required.

As it can often be difficult for investigators to produce 15-20 fertile males for embryo cryopreservation, we also offer the option of an IVF-Cryopreservation procedure. For this procedure, we will require one or two optimally fertile males. The males will be sacrificed to obtain sperm that is then mixed with oocytes in vitro. Two-cell embryos resulting will be vitrified and stored for future use. While this procedure is much easier on the investigator, there will often be long waiting times for this service due to the amount of technical time required. As in the embryo cryopreservation procedure, we will archive 200 embryos from a heterozygous x wild-type mating and 100 embryos from a homozygous x wild-type mating. All procedures will be tested by thawing a small aliquot of embryos and determining survival.

Injection of investigator-provided DNA construct into pronuclear stage mouse embryos/Service Request form plus documentation.

Policies:

Recombinant DNA Experiments

Because pronuclear injections and ES cell electroporations utilize unique DNA sequences that recombine into the mouse genome, the principal investigator needs to provide the GEM Facility with an approval number from the Institutional Biosafety Committee (IBC) prior to beginning these experiments.

Pronuclear Injection

Our facility has produced thousands of founder lines by pronuclear injection. While DNA can generally be shown to incorporate into the genome, the gene may or may not exhibit expression. It is not always possible to predict whether the transgene will be expressed and the effects of its expression in the mouse. For this reason, the GEM Facility does not guarantee transgene expression. The investigator is responsible for demonstrating a reliable scheme for identifying detection of the transgene at a single copy level by providing results from pilot experiments for this purpose. The GEMF will provide protocols for single-copy detection upon request.

Generation, isolation and implantation of mouse embryos into pseudopregnant females/Service Request form.

Isolation and cryopreservation of mouse sperm.  The GEMF will cryopreserve sperm from at least 2 males of prime breeding age.  Investigators will be requested to set up a timed mating just prior to the procedure (on a timeline designated by the technician) in order to insure the production of fresh sperm. All sperm cryo procedures are validated by subsequent IVF and culture to 2-cell.

Policy:

The background strain of the mice for freezing plays an important role in the success of the procedure. We may not be able to perform some procedures in certain strains of mice. Therefore, this information is critical for us to determine the potential success of your project.

Electroporation of investigator-provided construct with 10-15 colonies picked for analysis/Service Request form.

Pricing is available upon request, after consultation with the investigator to determine the requirements of the specific project.