Frequently Asked Questions
Imports and Exports
CRISPR / Cas9 Genome Editing
Step 1 Obtain a Purchase Order (if required by your Institute). Not required for Garvan researchers.
Step 2 Complete the Rederivation service order on StuartWeb.
Step 3 ABR staff will contact you to discuss the rederivation process.
Imports and Exports
Step 1 Obtain approval from the owner of the mouse line and check if a Material Transfer Agreement (MTA) or other approval is needed.
Step 2 Obtain relevant Animal Ethics (AEC) Approval and Institutional BioSafety (IBC) Approval.
Step 3 Obtain a Purchase Order (if required by your Institute). Not required for Garvan researchers.
Step 4 Complete the Request to Import Live Mice service order or the Request to Import Frozen Embryo Sperm service order on StuartWeb.
Step 5 The ABR Imports/ Exports Co-ordinator will:
- Contact the donor facility/ place a formal order
- Obtain an import permit
- Request health screens that will be assessed by the ABR veterinarian
- Advise if rederivation of the mice will be required for breeding at ABR
- Organise the courier
Step 6 If rederivation is required complete a Rederivation Request service order by selecting 'Rederive' in the 'Plans for Mice' section of the Import service order on StuartWeb.
The transport of frozen sperm and embryos is becoming more common and does prevent any risk to animal welfare during transport especially on long international flights. Australian Quarantine now allows the import of both frozen mouse sperm and embryos under some very strict conditions. Before making a decision the following factors should be taken into account:
- The donor facility must be able to meet the exact requirements of the import permit to prevent problems on arrival in Australia. Any failure to meet the conditions may result in the imported sperm / embryos being destroyed on arrival.
- The donor facility must have experience in reliably freezing mouse sperm and embryos and must provide their thawing protocol.
- The cost of transporting frozen sperm or embryos is similar to the cost of importing live mice plus rederivation.
- Imported sperm and embryos can generate a clean mouse line on arrival without the need for additional rederivation.
- The cost of transporting live mice from a clean facility and performing thorough health screening on arrival is still cheaper than importing frozen sperm and embryos.
Step 1 Ensure you have approval to transfer the mice to another institute. The recipient may need to sign a Material Transfer Agreement (MTA) or obtain other licensing approval.
Step 2 Obtain a Purchase Order (if required by your Institute). Not required for Garvan researchers.
Step 3 Complete the Request to Export Live Mice service order on StuartWeb.
Step 4 The ABR Imports/ Exports Co-ordinator will:
- Contact the recipient facility to obtain permission to send the mice
- Send the ABR health screen to the recipient facility
- Organise the courier
- Advise Australian Quarantine of the upcoming export (international exports only)
- Organise the vet check for Australian Quarantine (international exports only)
- Organise paperwork including customs declaration and export permit for international exports.
Step 5 Mice will be sent accompanied by a Mouse Passport - a report providing information on genotypes, birth dates, sex, phenotype and line production statistics.
Step 1 Ask the relevant research group if they are willing to provide mice.
Step 2 Sign a Material Transfer Agreement ( MTA) or obtain other licensing approval if required.
Step 3 Obtain AEC and IBC approval. For Garvan/ St Vincent precinct this includes:
- Adding the line to an existing protocol using the Inclusion of New Lines Form (AEC intranet) or submitting a new protocol application including the new line.
- A current IBC approval covering the work. Contact the relevant AEC or IBC for further details.
Step 4 Ask the owner of the mice to complete the relevant Transfers service order on StuartWeb.
NOTE: For transfers within the Garvan/St Vincent precinct the form must be sent to the AEC executive, and animal facility manager and the Transfer between Garvan researchers form is used. For transfers between ABR partners or ABR partners and Garvan researchers the Transfer between ABR partners or between ABR partner/Garvan service order is used.
Step 1 Complete either the Sperm Freezing service order, or the Embryo Freezing service order on StuartWeb.
Step 2 ABR staff will update the service order with milestones and comments in the StuartWeb platform.
NOTE: If you wish to import live mice for cryopreservation complete an Import Live Mice service order on StuartWeb and indicate the Cryopreservation request in the 'Plans for mice' section of the order.
Sperm freezing is more commonly used because it has the following advantages:
- Only requires 4 males
- Can be completed quickly- usually within 4-6 weeks depending on the method of validation chosen
Embryo freezing is only preferred if:
- The line is on a mixed background that you want to preserve
- Male mice carrying the gene of interest have fertility problems
- Sperm freezing requires 4 sexually mature males at 10 weeks or older.
- Validation demonstrates that the line has been successfully frozen and can be recovered.
- The “No validation” option still involves thawing one vial of frozen sperm and checking that the sperm show normal motility. This does not guarantee that the sperm are able to fertilise an oocyte.
- While validation using IVF and culture to blastocyst embryos demonstrates that the sperm is capable of fertilising mouse ova, the recovery of live pups demonstrates the subsequent viability of the embryos and is the ultimate validation test.
- The cost differential between the different validation methods reflects the work involved.
- While it is very rare to have difficulties in recovering a cryopreserved line it is recommended that any line with known fertility problems or with aged donor mice should be validated to either blastocyst embryo or live pups.
Storage is split between the ABR and Garvan Darlinghurst cryostorage sites. This provides security in unlikely event of damage to either cryostorage facility.
CRISPR / Cas9 Genome Editing
1. How long does it take to produce a new GM mouse line using CRISPR/Cas9?
The time taken will depend to a large extent on the complexity of the modification and whether developmental phenotypes are associated with inactivation of the targeted gene.
In the absence of these adverse phenotypes, production and identification of founder mice from Level 1 Projects (Gene KO or larger deletions) can take as little as 8-12 weeks from placement of the order.
The minimum time for Level 2 Projects is 10-14 weeks due to the synthesis of targeting oligonucleotide Longer project timelines (up to double the minimum) can occur in periods of high demand, or with suboptimal targeting sites (eg AT-rich).
Level 3 Projects involve production of a targeting plasmid and a less efficient rate of homologous targeting. For these projects a miniumum time of 5-6 months is expected with more difficult or complex projects requiring as long as 9-12 months.
2. Why are there three Project Levels?
Gene targeting using CRISPR/Cas9 in mice can be divided into three basic approaches that generate the desired founders with decreasing levels of efficiency and therefore increasing cost:
Level 1 ($8,054) - targeting one or two sgRNAs to a specific locus to introduce small (1-20bp) insertions/deletions or larger deletions (0.1-5.0kb).
Level 2 ($10,715) - incorporating small changes (e.g. point mutations, small (1-60bp) specific insertions/deletions) via gene targeting with a sgRNA and homologous recombination with a single-stranded oligonucleotide DNA substrate (~150 bases).
Level 3 ($16,110) - coupling of the targeting of one or two sgRNAs to a specific locus with subsequent homologous recombination with a plasmid DNA substrate (5-10kb) to either "flox" an exon or "knock-in" a reporter or other large piece of sequence.
3. How are the founder mice identified and what sort of information do you provide to the client about sequence results?
Founder mice are identified by Sanger sequencing of PCR amplicons derived from the targeted locus. The precise sequence of the modified locus will be provided to the client indicating all changes from the wild-type sequence.
The screening protocol used to obtain and sequence PCR amplicons will be made available to clients. The Garvan Molecular Genetics (GMG) facility is also available to clients to develop an economical and automated high-throughput screen based on DNA melt-curve analysis.
4. How do you minimise off target effects?
Selection of optimal single guide RNA (sgRNA) sequences is performed using the algorithm developed by the Zhang laboratory (MIT) which allows identification of sgRNAs with the lowest potential for off-target modifications. Whilst every effort is made to avoid off-target modifications, MEGA cannot guarantee that they will not be present in founder mice. A list of potential off-target sites can be provided to the client for them to analyse if they wish. Even if present, off-target modifications are extremely unlikely to be linked to the desired mutation and can be removed by backcrossing to wild-type mice for 2-3 generations.
5. Can multiple genes be modified at the same time and does this impact on efficiency or make the process longer?
Simultaneous modification of up to four genes has been performed in Level 1 Projects (Gene KO or larger deletions) and is offered for a cost of $8,054 plus $1,500 for each additional targeted gene. Inactivation of multiple genes results in only a minor extension of the Project timeline (2-3 weeks) and is a rapid and cost-effective way to obtain gene KOs instead of importing, re-deriving and cross breeding of existing GM mouse lines.
6. What happens if there are no births or founders from the injection sessions?
An absence of births and/or founders often means that inactivation of the targeted gene results in embryonic lethality. The likelihood of this occurring will be discussed with the client before the Project commences. If no founders have been obtained after a reasonable period the MEGA team will contact the client to discuss project termination. The upfront payment of 50% of the total project cost is not refunded on project termination.
8. Can MEGA cryopreserve mice as part of the service?
Yes, Cryopreservation of a line using sperm freezing can be performed for an additional cost. Freezing can be done when 4 male mice of the correct genotype are no longer required for breeding or experimentation and can be sacrificed for sperm freezing.
9. Are the new lines produced on a clean background so I can easily transfer the mice to my facility?
Yes. The microinjected embryos are transferred into super clean recipient female mice to ensure they are pathogen free on delivery. Health reports will be provided to the recipient facility.
10. Why do I need AEC and IBC approval if ABR already has those approvals in place?
ABR has an Animal Suppliers license and can only send mice to other facilities if the recipient has current Animal Ethics Committee (AEC) approval covering the new line of mice. In addition the ABR facility is a PC2 animal facility and must maintain records regarding the Institutional BioSafety Committee (IBC) approval that covers the use of the new GM mouse line.