Mouse embryonic stem cells (mESCs) are an important tool for the investigation of pluripotency and lineage analysis.

Deriving mouse embryonic stem cells (mESCs) from genetic models provides an important tool for the analysis of the effect of a genetic change on development. It can also provide a tool for additional genetic manipulation.

What we will do

Embryos will be collected from the model of interest at E3.5, and at E4.5, epiblasts will be collected from late stage blastocysts and transferred to culture.

Each embryo will be treated separately and a small portion of successfully derived lines will be provided to the investigator for genotyping.

Up to 4 ES cell lines will be expanded and three vials of each will be frozen and supplied to the investigator.

What you need to supply

• Three female mouse for breeding (and the mutant breeder male).
• Demonstration that an assay is in place for genotyping.

Special considerations

The investigator will need to perform the genotyping within 24 hours of receiving samples.

Creating Mouse Embryonic Fibroblasts (MEFs) allows investigators to establish a primary culture from genetically modified animals.

What we will do

MEFs are generated from E13.5 or E14.5 dcp (day post-coitum) embryos. Cells may be obtained from an earlier stage of development if homozygosity of a line is lethal before E13.5, although the yield of fibroblasts will be reduced.

MEFs are then expanded and frozen in liquid nitrogen indefinitely for future use.

Known genotypes

If the embryos are of known genotype (i.e. both parents are homozygous at the locus of interest), we will extract MEFs from up to three embryos, culture and harvest the cells. Up to five aliquots of 3 x 106 per cryo-vial will be frozen. A “thaw test” will be performed before transferring the cells to the requestor.

Unknown genotypes

If the embryos are of unknown genotype (i.e. parents are heterozygous), we will extract MEFs from up to 10 embryos. The requester will be asked to genotype a small sample from each embryo within 24 hours, and we will continue to culture up to three embryos of each desired genotype.

What you need to supply

• Female mouse/mice
• Male breeder from which to establish a MEF cell line

Special considerations

The animals used for this procedure will be assigned to the facility transgenic protocol, so investigators do not need to write a protocol for this procedure or to cover these animal numbers.

Harvesting of embryos can be performed for a variety of applications.

What we will do

Embryos will be collected from the timed-pregnant mice and treated as requested by the investigator. The embryos can be collected at different stages from timed-pregnant female mice.

What you need to supply

• Female mice for generating timed-pregnancies
• Solutions and instructions for embryo processing
• An animal protocol for the timed pregnant females

Special considerations

Investigators should be aware that the number of embryos present in a timed pregnant mouse is highly variable.

Diploid embryo/ES cell aggregation can generate a mouse chimeric for a genetic alteration of interest.

What we will do

Genetically modified mouse embryonic stem cells (mESCs) are aggregated with diploid CD1 embryos to generate chimeric mice. The ES cell line will be expanded and the cells will be aggregated with a minimum of 150 CD1 embryos.

The aggregated embryos will be transferred into CD1 pseudo-pregnant females.

Offspring will be monitored for chimeras. At weaning, chimeras can either be transferred to the investigator’s protocol for testing of germ line transmission or maintained for up to two weeks at the investigator’s expense.

What you need to supply

• The ES cell line with evidence of mycoplasma testing (chromosome counting is also recommended)
• Evidence that a screening procedure has been planned to identify the transgene
• An animal protocol is required for the transfer of the founding mice

Special considerations

Please note that the facility cannot guarantee germline transmission of different mouse ES cells and chimeras.

Blastocyst injection of genetically altered mouse ES cells is used to generate chimeric mice carrying the genetic alteration(s).

What we will do

ES cells will be expanded for microinjection and injected into 20-30 albino B6 blastocysts. They will then be transferred into two to three CD1 pseudo-pregnant females.

Offspring will be monitored for chimeras and at weaning, transferred to the investigator for testing of germ line transmission. They can also be maintained for up to two weeks at the investigator’s expense.

What you need to supply

• The ES cell line with evidence of mycoplasma testing (chromosome counting is also recommended)
• Evidence that a screening procedure has been planned to identify the transgene
• An animal protocol is required as soon as there is evidence that germline transmission has been obtained

Special considerations

Please note that the facility cannot guarantee germline transmission of different mouse ES cells and chimeras.

CRISPR/Cas9 allows precise edits to be made in order to create knock-out, knock-in or conditional mouse models.

What we will do

After meeting with the investigator, a design for the model will be created.

Once the design has been approved by the investigator, the reagents (guide RNA and ss Donor if applicable) will be ordered.

The editing efficiency of the reagents will be tested in zygotes prepared by in vitro fertilization. If editing efficiency is acceptable, zygotes will be electorporated and transferred to pseudopregnant female mice.

After weaning, the founders will be transferred to the investigator's protocol or maintained for up to two weeks at the investigator's expense.

What you need to supply

Female and male mice will need to be provided if the gene edits are to be made on a mutant background. The facility will supply wild type C57BL6 or CD1 mice.

Special considerations

The investigator must prepare an animal protocol to receive the founder mice. The facility cannot guarantee that the targeted gene will be successfully altered or transmitted in the germ line.

Gene targeting vectors or expression vectors can be introduced into ES cells by electroporation and selected for subsequent screening and aggregation.

Most gene targeting is now done using CRISPR/Cas9. For specialized models that require vector construction please contact us directly to discuss the approach and pricing.

What we will do

We will meet with the investigator to determine the scope of the project. Then, strategy and workflow will be planned individually for each project.

What you need to supply

The role of the facility and investigator will  be optimized for each project.

Special considerations

This approach is no longer used routinely by our facility as this approach has been largely replaced by CRISPR/Cas9 mediated modification of mouse genes.

Pronuclear injection allows for the introduction of DNA or RNA for expression in the genome.

What we will do

Pronuclear injection is commonly used to inject genetic material directly into the nucleus of a fertilized oocyte. Fertilized eggs are collected at E0.5 when they are a single cell. Linearized DNA, RNA and/or protein, as appropriate, is injected into one of the pronuclei. The injected oocytes are then transferred into a pseudo pregnant mouse.

We will inject the DNA into a minimum of minimum of 150 oocytes and implant them back into female mice.

Founders (minimum of three) will be monitored. At three weeks, tail biopsies will be obtained and provided to the investigator for genotyping.

After weaning, the founders will be transferred to the investigator for testing germline transmission.

Before you begin

Before contacting us, please have the following in place:

• Justification to generate the model
• An animal protocol to receive the founder mice
• A genotyping assay for the transgene 

What you need to supply

• DNA for injection. Please consult the facility for a protocol for preparation of the DNA for injection. If required, the facility can prepare, quantify, and digest the construct, and prepare the insert for injection.
• Three ug DNA at 100 ng/ul. We will dilute this in injection buffer to a concentration of 50 ng/ul. 
• A picture verifying the quality, quantity and complete digestion of the DNA to be injected.

Special considerations

Please note that the facility cannot guarantee that the transgene will be expressed or transmitted in the germ line.

Embryo freezing allows investigators to permanently store mouse lines.

The investigator should be aware that the number of embryos that can be collected from these methods varies significantly among strains. Although the facility attempts to store at least 100 embryos per line, this cannot be guaranteed.

What we will do

Prepare medium for thawing of the embryos based on the protocol supplied by the site that froze the embryos.

Thawed embryos will be transferred to pseudopregnant females.

The mice will be monitored until delivery and samples from the offspring will be provided at weaning to the investigator for genotyping

Pups with the desired genotype will be transferred to the investigator’s protocol.

What you need to supply

• Frozen embryos or female/male mice to use to generate embryos
• Confirmation of the availability of an assay to genotype the offspring
• An animal protocol to accept the offspring

Special considerations

The facility cannot guarantee the success of an embryo transfer. If the investigator is ordering embryos from an external source, assuring that a control embryo vial is provided by the vendor can increase the likelihood of success. The control embryos will be used to optimize conditions.

Ovary freezing allows investigators to permanently store mouse lines. It also allows investigators to “rescue” transgenic lines that are difficult to propagate.

What we will do

When the ovaries are collected, they are cut in half and either frozen in a cryovial or transferred to a histocompatible female.

If the ovary is transferred to another female, the mouse will be transferred to the investigator for testing the viability of the transferred ovary.

The animal protocol for the mice and procedure, as well as testing of the ovary viability, is covered by the Central Animal Care breeding protocol until successful transmission of the genetic alteration of interest is demonstrated.

At that time, the investigator will need to obtain an approved animal protocol.

What you need to supply

• Female mice at four to eight weeks of age from which to generate the ovaries.
• Female mice for implanting the ovaries (histocompatible female mice).

Storage

The frozen ovaries can be transferred to the investigator for storage or stored in the facility.

Special considerations

• Female donor mice will be sacrificed.
• The facility cannot guarantee the success of the ovary transplantation. 
• Investigators are responsible for all housing costs.

Sperm freezing allows investigators to permanently store mouse lines.

What we will do

Sperm is collected from male mice demonstrated to be fertile. Sperm will be collected and frozen in aliquots of 150 ul x 6 cryovials. One vial will be thawed and the sperm motility tested.

What you need to supply

• Two proven-fertile male mice.

Storage

The frozen sperm may be transferred to the investigator for storage or stored in the Facility (in two different storage places).

Special considerations

The male mouse is sacrificed for the collection of sperm.

What we will do

Prepare medium for thawing of the sperm based on the protocol supplied by the provider.

Fresh or frozen mouse sperm will be used for in vitro fertilization of mouse oocytes collected from superovulated female mice. Fertilized oocytes will be transferred to pseudopregnant females.

The mice will be monitored until delivery and samples from the offspring will be provided at weaning to the investigator for genotyping. Pups with the desired genotype will be transferred to the investigator’s protocol.

What you need to supply

• Frozen sperm or male mice that are proven to be fertile (for sperm collection)
• Confirmation of the availability of an assay to genotype the offspring
• An animal protocol to accept the offspring

Special considerations

The facility cannot guarantee the success of an in vitro fertilization. If the investigator is ordering sperm from an external source, assuring that a control sperm sample is provided by the vendor is important to increase the likelihood of success. This control sperm sample will be used to optimize the procedure.