Transgenic Core Services

The conventional and traditional method for creating gene targeted mice was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts (see ES cell Microinjection). This work is extremely laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted, as a faster and cheaper method for creating gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once a cut has been made it is possible to:; insert, delete, or alter a specific sequence of interest.

Microinjecting Cas9 mRNA into zygotes is the preferred method for large gene sequence modifications or conditionals mutations. While some labs may prefer to prepare their own CRISPR reagents, we can also recommend utilizing the Penn School of Medicine CRISPR/Cas9 Mouse Targeting Core or ordering the guides through a commercial vendor such as IDT.

While success rates may vary for each gene target region, there have been no lines that we were unable to generate founder animals. The average success rate for microinjections is 30%.

Your mRNA will be injected directly into the cytoplasm of a minimum of 150 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type strain, or existing mutant strain. The mRNA is loaded into the ultra-fine injection needle, and using a constant, regulated flow of pressure is injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for integration of the mRNA sequence. Mice identified with the correct sequence are considered the founders, or F0, these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid strains tend to work better than inbred strains.

If the core does not generate any founders from the first microinjection session a second session will be scheduled, the fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If no founders are identified from the second set of microinjections we will need to arrange a meeting to evaluate the project.

The conventional and traditional method for creating gene targeted mice was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts (see ES cell Microinjection). This work is extremely laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted, as a faster and cheaper method for create gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once a cut has been made it is possible to:; insert, delete, or alter a specific sequence of interest.

Electroporation of the Cas9 RNP is typically employed for all mutations such as small insertions, deletions or sequence changes. While some labs may prefer to prepare their own CRISPR reagents, we can also recommend ordering the guides through a commercial vendor such as IDT.

The RNP will be electroporated with 0.5day gestation zygotes. The electroporation is performed using a Gene Pulser Electroporator and 1mm cuvettes. This procedure allows the RNP complex to pass through the chemically thinned zona pellucida, and penetrate the fertilized zygote where it incorporates into the mouse genome. Shortly after electroporation the zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for integration of the RNP sequence. Mice identified with the correct sequence are considered the founders, or F0, these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid strains tend to work better than inbred strains. A minimum of 90 electroporated zygotes are transferred into recipient mother mice, the majority of which will become pregnant. 

A cost saving can be offered if 2 lines are generated during the same electroporation session. Both lines can be for your own lab, or you can split the service fee with another investigator.

If the core does not generate any founders from the first electroporation session a second session will be scheduled, the fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If no founders are identified from the second set of electroporations we will need to arrange a meeting to evaluate the project.

Your DNA will be injected directly into the pronucleus of a minimum of 150 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type strain, or existing mutant strain. The DNA is loaded into the ultra-fine injection needle, and using a constant, regulated flow of pressure is injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for integration of the exogenous DNA sequence. Mice identified with the correct sequence are considered the founders, or F0, these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

The facility will inject your DNA construct on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid strains tend to work better than inbred strains. A minimum of 100 microinjected zygotes are transferred into recipient mother mice, the majority of which will become pregnant.

We expect to generate 30-50 pups from a microinjection session, this number is typically sufficient for the production of founder mice. Although it is common that founder mice can be generated from fewer animals too.

If the core does not generate any founders from the first microinjection session a second session will be scheduled, the cost of the additional animals will be billed to you, but the core will absorb the service fee. If, however, no founders are identified from the second set of microinjections we will need to arrange a meeting with the principle investigator to discuss the project.

PREPARATION OF TRANSGENE DNA FOR MICROINJECTION
General Considerations

DNA samples for microinjection should be free of contaminants that might harm the single cell zygotes. Such potential contaminants include traces of phenol, ethanol or enzymes. It is also essential to remove any particles that could clog the injection needles. When purifying the DNA, please use powder-free gloves to avoid particulate matter that may interfere with the microinjection. Be aware that autoclaved tubes can have residue in them- ; use sterile cryovials as an alternative.

The Transgenic Core Facility recommends that all solutions used to prepare DNA, be filtered through a 0.2micron filter. Sterile, embryo-tested water (Sigma W-1503, 500ml) or Milli-Q should be used in all solutions.

Once you have isolated your injection construct and diluted it to the correct concentration for injection, a final pass through a 0.2micron syringe filter (Millipore, Cat. No. SLGVL04) will remove any remaining impurities that may clog the injection needle. This step is essential.

DNA Concentration
Submit a gel to verify the purity and determine the DNA concentration. Supply 10µg of DNA adjusted to a final concentration of 100ng/µL. The core will dilute the DNA to 1-3ng/uL for microinjection. A higher concentration of DNA will not necessarily increase the percent incorporation of your transgene; this could in fact cause developmental arrest of the embryos. Too low a concentration decreases the number of transgenic founders. Most constructs are injected in the range of 1.5-2.5ng/uL; when possible, approximately 2 Pico liters are injected into each embryo.

Procedure
The Qiagen QIAquickuiaquick extraction kit is a simple and fast way to obtain microinjection-quality DNA. Vector sequences must be previously eliminated by restriction digestion, since plasmidic DNA can significantly alter the expression of transgenes and is toxic for the ovum. Preparing the DNA involves the following steps:

• Perform a restriction digest to liberate the transgene from vector sequences. Final yield should be 10-20µg of transgene insert.
  Note: If the size of the transgene is close to the size of the vector, add restriction enzyme(s) to cut the vector in smaller fragments.
• Separate restriction digest products on a 0.8% agarose/TAE gel without EtBr. Use either low- or standard-melting temperature agarose.
• Stain the gel in a 1µg/mL EtBr solution in the dark and visualize the bands with long wavelength UV light. Minimize exposure time to prevent photochemical damage. Use a clean scalpel to cut out the band of interest. Remove as much excess agarose as possible.
• Transfer gel slice to a pre-weighted DNase free 1.5mL tube and proceed to DNA extraction-purification using the Qiagen Quiaquick QIAquick kit (column purification). In the last step of this procedure, use the injection buffer* to elute the DNA from the column.
• Load different DNA amounts (e.g. 1 to 5 µL) consecutively onper different slots of a 0.8% agarose/TAE gel against a standard to estimate the concentration and the purity.
• *Injection Buffer: 10mM Tris, pH 7.4; 0.1mM EDTA, pH 8.0

A picture of the gel and the concentration must be supplied to the technical director when submitting the request form.
Store the DNA at -20°C until needed 

Alternative Procedure
Purification of the DNA fragment using electro elution, followed by Elutip mini-columns.
Note: Never apply negative pressure to the Elutip..

1. To elute the fragment, prepare dialysis membrane by boiling appropriately sized strip in 95mL H₂O + 5mL 0.5M EDTA.
2. Digest 20-30µg of plasmid to separate the fragment from the vector backbone.
3. Use 0.7% agarose gel made in 1X TAE.
4. Excise the band (under long wave UV light) from the gel; insert gel fragment into dialysis bag.
5. Run gel at 100V for 10 minutes or as long as necessary to cause the DNA to exit the gel slice and line up on the dialysis membrane.
6. Pipette DNA and transfer to a 1.5mL tube. You can check the gel piece and the DNA solution under long-wave UV light.
7. Chill DNA on ice for 5 minutes; spin in a micro centrifuge for 5 minutes to remove any agarose.
8. Transfer electro elution solution to a new tube.
9. For the final clean up, adjust the NaCl concentration to 0.2M with 5M NaCl.
10. Prepare the column by running through 3mL of high-salt buffer, then 3mL of low-salt buffer by attaching the column to 3mL syringes
11. Run DNA sample through the column at about 1 drip/second using a 3mL syringe. The DNA binds to the column.
12. Wash the bound DNA using 3mL of low-salt buffer (1 drip/second).
13. Elute the DNA into a 1.5mL tube using 400µL of high-salt buffer in a 1mL syringe (1 drip/second).
14. Add 1mL 100% ethanol mix and precipitate overnight at -20°C.
15. Spin down the DNA for 15 minutes and re-suspend to final concentration.

Knockout mouse models are widely used to study human diseases caused by the loss of gene function. Examples of these diseases include cystic fibrosis, beta-thalassemia and various forms of cancer. Information from studies with knockout mice can lead to a better understanding of the pathogenic mechanisms of human genetic and infectious disease, as well as provide animal models that can be used to test new genetic and drug therapies to treat these disorders.

This artificially induced mutation is carried in every cell of a knockout mouse throughout development. The resulting phenotype (appearance, biochemical characteristics, behavior, etc.) may provide some indication of a gene's normal role in the mouse.

Knockout mice are produced by a technique called gene targeting. This technique works by isolating and activating a gene sequence and then replacing it with a version of the same gene sequence that contains a mutation. The replacement occurs by homologous recombination, where two very similar DNA sequences line up next to each other and exchange parts. Gene targeting is carried out in mouse embryonic stem (ES) cells. Embryonic stem cells are derived from very early (usually male) mouse embryos and have the capacity to contribute to the complete development of the animal. The aim is to get modified ES cells to contribute to a germ line. Some sperm are produced that carry the desired mutation and; these will then fertilize a wild-type egg. The resulting progeny develop with one copy of the mutated gene in every cell. Interbreeding these offspring will generate some homozygous individuals that carry both copies of the mutated gene – these are the knockout mice.

Prior to initiating chimera production, investigators must ensure they have provided documented evidence that the parental cell line has been screened for absence of microorganism contamination. It is important to remember that feeder cells (primary mouse embryonic fibroblasts, MEF) must also be screened for the presence of pathogens; these also have the potential to transmit pathogens to the ES cells. Additionally, the clones, containing the correct target sequence will need to be karyotyped to ensure they contain the correct number of chromosomes, to ensure they are not euploid.

(A) Normal Session: Chimeric mice will be produced by injecting Embryonic Stem (ES) Cell clones, containing a specific gene targeted mutation, into the blastocyst cavity of E3.5day embryos. The injected blastocysts are cultured 1-3 hours and then surgically transferred into the uterus of a 2.5d.p.c. pseudo-pregnant recipient mother mouse. Approximately 10 days after birth the pups are identified for level of cell contribution, which is apparent by the coat color differences. The fur of chimeric heterozygous pups will be a mix of the host blastocyst strain and the ES cell strain. Mice with the highest amount of cell-derived fur will be selected for future breeding to establish the colony. These mice will be transferred to you animal holding room, where they can be bred to check for germline transmission and to expand the line for experimental use.

(B) No ES Cells: Occasions may arise when the ES cells being, prepared for microinjection, are not viable, either due to contamination or growth-related problems. In this instance a lower fee has been developed, which will cover the cost of the work performed up-to the day of the microinjections. If the project needs to be repeated you will incur the full-service fee.

(C) Debris with ES cells: Often when the Transgenic Core receives ES cells, for microinjection, they contain lots of debris, either from dead cells or feeder cells. When this occurs, the debris obstructs access to the viable ES cells, sticks to the outside of the injection needle and will also clog the injection needle. These problems lead to a much lengthier microinjection day and also the use of more, than typical, injection needles. This additional timing and extra supplies are reflected in the increased fee structure for this service. The above problems do not inhibit the project from being completed, they just slow the process down.

The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any appropriate strain background requests. The core will microinject approximately 40-50  blastocysts for each ES cell line. Approximately 8-15 ES cells are microinjected into each blastocoel cavity. If available the transgenic core will microinject up to 2 clones of the same targeted line per session.

We expect to generate 10-20 pups from an ES cell microinjection session, this number is typically sufficient for the production of chimeric mice. Although it is common that founder mice can be generated from fewer animals too. Germ line transmission of chimeric animals cannot be guaranteed.

If the core does not generate any founders from the first microinjection session a second session will be scheduled, the fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If, however, no founders are identified from the second set of microinjections we will need to arrange a meeting with the principle investigator to evaluate the project.

ES CELL PREPARATION
General Considerations

• Established germ line-transmitting ES cells can be purchased from commercial sources, including: EMMA, MMRRC, Taconic, Specialty Media, Andras Nagy and Elizabeth Robertson (Harvard University).
• Variable coat colors may occur when using 129 derived ES cell lines. If considering the use of C57BL/6 ES cells, bear in mind that they are less stable in terms of maintaining germ line potential.
• ES cell lines and feeder cells should be tested for the presence of pathogens. Mycoplasma is the most common pathogen found in ES cells.
• Culture the ES cells at a reasonably high density, split at 1:3 to 1:6 every 2-3 days.
• Freeze vials of stock ES cells to minimize chromosomal abnormalities. Keep records of the number of passages after thawing. Always return to the original stock for each experiment.
• Do not grow cells for more than 3 or 4 passages before beginning an experiment. It is important to reduce the time they spend in culture before introduction to the blastocyst.
• Thaw cells 3-4 days prior to the scheduled microinjection date.
• Chimeras should not be bred until they are sexually mature or at least 7-8 weeks old. They should be bred to the same strain as the host blastocyst. Occasionally, several litters need to be generated before the desired genotype is produced. Female chimeras are not generally used.

Procedure
Note: Allow yourself at least 2.5 hours for preparing the ES cell for microinjection.

1. Change the ES cell media 2 hours before proceeding with the following steps.
2. Remove the medium from the flask and wash the cells with PBS (without Mg/Ca).
3. Add 2-3mL of 0.25% Trypsin, and incubate the cells for ~5 minutes at 37°C.
4. Stop the trypsin reaction by adding 4mL of ES cell culture medium. Re-suspend the cells by gently pipetting them up and down three or four times. Do this until the cells produce a single cell suspension. Do not pipette more than necessary.
5. Place the suspension in a gelatinized T.C. dish and incubate the cells at 37°C for ~30 minutes.
6. Most of the MEF feeders will be loosely attached to the plastic surface, whereas most of the ES cells will be in suspension. Move the dish very carefully, taking care not to disturb these layers. Very carefully tilt the dish and collect the entire medium containing the ES cells, leaving the MEFs attached to the dish.
7. Centrifuge the cell suspension at 200g for 5 minutes and remove the supernatant.
8. Re-suspend the cells thoroughly in 2ml of ES cell injection medium.
9. Place the tube on ice for 30 minutes.
10. Carefully remove the top 1/4 of the medium, taking care not to disturb the cells that have settled at the bottom of the tube. This step allows the removal of dead cells floating in the medium.
11. Transfer the middle portion to a sterile tube and place on ice. Label this tube ‘ES cells.’ The remaining tube should be labeled ‘Debris’. Both tubes are now ready to be delivered to the transgenic core staff.

Manipulating the Mouse Embryo; Nagy, Gertsenstein, Vintersten, Behringer, Cold Spring Harbor Laboratory Press, Fourth edition.

The conventional and traditional method for creating gene-targeted rats was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts. This work is laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted as a faster and cheaper method for creating gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once DNA has been cut it is possible to insert, delete, or alter a specific sequence of interest.

Microinjecting Cas9 mRNA into zygotes is the preferred method for large gene sequence modifications or conditionals mutations. While some labs may prefer to prepare their own CRISPR reagents, the Transgenic Core Lab also recommends using the Penn School of Medicine CRISPR/Cas9 Mouse Targeting Core or ordering the guides through a commercial vendor such as IDT. 

Your mRNA will be injected directly into the cytoplasm of a minimum of 100 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type or existing mutant strain. The mRNA is loaded into the ultra-fine injection needle and using a constant, regulated flow of pressure is then injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab or the CRISPR Core and screened for integration of the mRNA sequence. Rats identified with the correct sequence are considered the founders, or F0; these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

The Transgenic Core will schedule strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid/outbred strains tend to work better than inbred strains. A minimum of 50 microinjected zygotes are transferred into recipient mother rats, the majority of which will become pregnant.

If the core does not generate any founders from the first microinjection session a second session will be scheduled. The fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If no founders are identified from the second set of microinjections we will need to arrange a meeting to evaluate the project.

Mouse line rederivation is used in many institutions as a method of re-establishing you mouse line or as a means to eliminate pathogenic or potentially pathogenic agents. This procedure involves transferring pre-implantation stage zygotes into recipient surrogate mothers (for completion of development). Fertilized zygotes for rederivation can be generated in several different ways, described below:

1. –Harvest and freeze embryos; This option is performed for complex (more than 2 mutant alleles) mouse lines housed in ARC, that do not possess the required health status to enter the CTRB animal facility. Your males (minimum of 5 mice, 7 weeks old+) are mated with your females (5 mice, 3-5 weeks old), the resulting fertilized zygotes are harvested and cryopreserved. A day or two later the zygotes are thawed and transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use. 
2. -Pre-frozen embryos; The fastest and most efficient method for importing a mouse line is to have pre-frozen embryos shipped directly to the Transgenic Core. We will receive the shipment, store appropriately until needed and regenerate the mouse line. Alternatively, the strain may already be archived in our storage units. To recover the strain, vials are removed from liquid nitrogen, the zygotes are slowly thawed and rehydrated then the zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use. 
3. -Cooled sperm; This service is ideal for importing strains from institutions who do not have access to cryopreservation facilities. Reproductive tissues from 1-2 male mice are shipped to the Transgenic Core at 4 degrees. Upon receipt of the samples the Core will perform In-Vitro Fertilization (IVF) with oocytes isolated from wild-type donors. Fertilized zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use. This service requires a strict schedule, due to the viability of the sperm, and the essential timing involved when performing IVF.
4. -Pre-frozen sperm; The pre-frozen sperm can be imported directly to the Transgenic Core. We will receive the shipment, store appropriately until needed and regenerate the mouse-line. Alternatively, the strain may already be archived in our storage units. To recover the strain, vials are removed from liquid nitrogen, the sperm is slowly thawed, allowed to recover then used for IVF with oocytes, isolated from wild-type donors. The following day viable zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use. 
5. -Freeze and thaw sperm for IVF; This option is performed for less complex mouse lines housed in ARC, that do not possess the required health status to enter the CTRB animal facility. Tissues are removed from 1-3 males and the sperm is isolated and frozen. Approximately a week later a sample of the sperm is thawed and used for IVF with oocytes isolated from wild-type donors. Fertilized zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use. 

The method of rederivation you choose to adopt should be determined based on the genotype of the animals and your goals with the strain. Strains involving 2 or more allele modifications ideally should not be rederived using in-vitro fertilization options. The zygote transfer methods (A or B) would produce mice with the correct genotype much faster than the sperm options.

All strains of mice can be rederived, but the efficiency of the procedure depends on the hardiness and the fertility rate of the strain. Inbred strains may occasionally require the use of more mice and could take exceptionally more time.

After the zygotes have been surgically transferred into the reproductive tracts of the surrogate mother mice they will remain in the care of the transgenic core. The surrogates will deliver the pups 3 weeks post-transfer. Approximately 2 weeks later the Core will isolate tail snips for your lab to genotype. The offspring will be weaned when they are 3 weeks old. At this time the Veterinary Department may decide to perform some health assessments on the animals to verify their health status. This procedure will take approximately one to two weeks to complete. After which, the animals can be transferred to your animal holding room. 

Cryopreservation provides a useful tool for archiving mouse lines. The reasons to cryopreserve strains include:

• Protect against the loss of valuable strains through breeding failure, disease, human error, etc.
• Eliminate the cost of maintaining inactive mouse lines.
• Free up space for other mouse lines.
• Facilitate future rederivation into a new facility.

Frozen embryos can be stored indefinitely and thawed many years later, should the investigator require the use of the archived mouse line.

Archiving a complex, involves more than two mutant alleles, mouse line is accomplished by freezing a minimum of 250 embryos. This method necessitates the use of 10-20 of your young male mice mated, overnight, with 10-20 of your female mice. The following day, viable 2-cell embryos are isolated and cryopreserved. The straws, containing the frozen embryos, are held in the Core large storage unit until needed. Viability of your cryopreserved line is performed by thawing a sample of embryos and culturing to the blastocyst stage. If requested, we can also surgically transfer some of the thawed embryos to generate live mice, but this would involve additional service fees.

Investigators who require a strain to be frozen using embryos, are often confounded and inhibited by the number of mice needed to complete this task. To overcome this hurdle, we have the ability to perform a large IVF service. In doing so we are able to generate the desired number of embryos requested to secure the strain. This can be completed using wild-type oocyte donor females, or mutant oocyte donor females, and sperm from one of your males. If using your donor females, we would need ten young (3-5 weeks old) mice. Males supplied for this service should always be individually housed, healthy and a minimum of 12 weeks old.

Cryopreservation provides a useful tool for archiving mouse lines. The reasons to cryopreserve strains include:

• Protect against the loss of valuable strains through breeding failure, disease, human error, etc.
• Eliminate the cost of maintaining inactive mouse lines.
• Free up space for other mouse lines.
• Facilitate future rederivation into a new facility.

Frozen sperm can be stored indefinitely and thawed many years later, should the investigator require the use of the archived mouse line. The Core will cryopreserve 15 samples (straws) of sperm for each strain. To achieve this, you must supply 3 healthy male mice, minimum of 12 weeks old, the mice will need to be individually housed at least 4 days prior to the scheduled freeze date. The sperm from the males is pooled together for the freezing. If necessary we can freeze the sperm from each male separately, but this will incur additional fees. There are two options for freezing sperm: without in-vitro fertilization, or with in-vitro fertilization.

Sperm Cryopreservation (without IVF quality control)
Approximately 2 days post-sperm freezing, a sample is retrieved from the liquid nitrogen unit, it is thawed, incubated, and used to assess the viability of the strain. The viability of the line is recorded and relayed to your lab. If the viability of the sperm is extremely low, a second session of freezing will be required to safely secure the line. However, it is not often that this is needed.

Sperm Cryopreservation (with IVF quality control)
Approximately a week post-freezing a single straw of the sperm is used for IVF, with wild-type oocytes. The following day viable zygotes are transferred into surrogate mother mice to reestablish the mouse line. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype can be transferred to your animal holding room for future use, or euthanized. 

Frozen stocks of mouse embryos and sperm are stored in the Transgenic Core large storage units. Often investigators opt to share their unique mouse lines with researchers at other institutions. This service fee covers the time and supplies needed to remove the appropriate samples from the storage unit, coordinate the shipment and transport the samples to the Shipping Core Facility.

Importing frozen strains is a valuable resource utilized by investigators who need to use unique strains, that are not already housed in the CHOP facility. The external institution will ship the frozen sperm, or embryos, directly to the Transgenic Core. Upon receipt the Core will unpack and store the gametes for future use. The Core will also arrange return of the shipping container.