- How is the nomenclature of EUCOMM alleles?
- What are the EuMMCR unit handling fee prices?
- How long does it take to receive knockout resources from the EuMMCR unit?
- How is the order process structured?
- Are there cancellation fees?
- What Quality Control is included in EuMMCR clones?
- What quality control procedures are implemented by EUCOMM/the EuMMCR unit to ensure non contaminated ES cell clones?
- Are EUCOMM ES cells male or female?
- Which wt ES lines have been used to generate conditional knockout clones?
- Is the Ordering of wt ES cells used in the EUCOMM program possible?
- Are there any warnings and recommendations?
How is the nomenclature of EUCOMM alleles?
tm1a: The targeted allele in EUCOMM ES cells is "tm1a". ES cells are male and heterozygous for the targeted mutation. "tm1a" is a so called targeted trap allele: it has been generated by targeting, yet it functions as a gene-trap knockout.
In cases where the target gene is small and does not contain a "critical" exon, the approach is to split an exon and introduce the targeting cassette imbedded in an "artificial intron".
tm1b: This allele is a lacZ tagged null allele. The critical exon has been deleted using Cre recombinase.
tm1c: The conditional allele is called "tm1c". It is generated by crossing "tm1a"-mice to mice, which are ubiquitously or germline expressing Flp recombinase. "tm1c" is a functional wild -type allele. However, the so called critical exon (= targeted region) is flanked by loxP sites, which allows for excision of the critical exon with Cre recombinase.
tm1d: The organ or cell type specific null allele is called "tm1d". This null allele is generated by crossing "tm1c"-mice to a mouse that is expressing Cre recombinase in the desired organ or cell type specific manner. Cre recombinase will excise the critical exon, which for most EUCOMM alleles will cause a frameshift and loss of downstream protein sequence due to nonsense mutations.
tm1e: This allele is non-conditional because the downstream loxP site is missing. The 3′ loxP site is often lost due recombination events in the homology region between the targeting cassette and 3′ loxP site. Approximately one half of the clones retain the loxP site, however, in extreme cases, the loxP site is absent in all clones. These lacZ-tagged alleles report endogenous gene expression and are highly likely to be null mutations. However, these mutations cannot be converted to conditional alleles with Flp recombinase.
Examples of the EUCOMM nomenclature:
What are the EuMMCR unit handling fee prices?
The EuMMCR unit price structure is the following (since April 1st, 2015):
|1 ES cell line for one gene||EUR 1,600.00|
|2 ES cell lines for one gene||EUR 2,600.00|
|3 ES cell lines for one gene (recommended)||EUR 3,600.00|
|4 ES cell lines for one gene||EUR 4,600.00|
|5 ES cell lines for one gene||EUR 5,600.00|
|One additional vial of an ES cell clone||EUR 200.00|
|Chromosome counting (per clone)||EUR 600.00|
|Targeting vectors (intermediate or final)||EUR 750.00|
|Vector cassette||EUR 300.00|
|Vector backbone||EUR 300.00|
|Wild Type ES Cells|
|Wildtype ES cells (e.g. JM8.N4 or JM8A3)||EUR 1,000.00|
The handling fee does not include shipping. The EuMMCR unit requests from users their FedEx or (in Germany) TNT account numbers and uses this number for shipment.
How long does it take to receive knockout resources from the EuMMCR unit?
ES cells: Upon user request the EuMMCR unit thaws, expands and re-freezes several aliquots of the requested clone(s) in order to be able to provide the user with one aliquot and sustain the resource.
ES cell culture and the quality control means up to 6 month lab work for us. From the lab's point of view, the EuMMCR unit is able to send the ES cell clones out after 3 - 4 month most of the times.
Targeting Vectors: Upon request the EuMMCR unit prepares DNA of targeting vectors, starting from the glycerol cultures we received from the EUCOMM vector production lab. For quality control we do restriction mapping on the vectors. This also will take between 4-6 weeks.
How is the order process structured?
There are a few steps to take until you receive the ordered probes.
Here is a rough summary of how this will work:
- We will start expanding the requested ES cells or vectors immediately after receiving your order and perform our well-established quality controls on vectors and ES cell clones.
- You will receive an email with a Material Transfer Agreement (MTA). Please print two copies, sign them and send both to our legal affairs department.
- We will countersign the MTA and send one copy back to you, together with the invoice.
- When we receive your payment, the material is released by the legal affairs department.
- If the QC is finished, we will contact you to announce the shipment.
- We will dispatch the material to you.
Are there cancellation fees?
To ensure delivery of the requested material in a timely manner (usually 3-4 months according to our production timeline), we will start with the production of the material immediately after the order has been placed.
The order can be cancelled free of charge within 14 days after the order date, i.e. after the receipt of the order confirmation email. To cancel your order within this period please send an email to .
Once the 14-day period has passed, a cancellation fee of 30 % of the total payment amount will be charged. Should the material be ready for shipment by the time you wish to cancel the order, there will be no refund.
Thank you for your understanding.
What Quality Control is included in EuMMCR clones?
We only send out ES cells that pass all our well-established quality control tests.
Some QC tests are already applied on the ES cells in the production lab. EuMMCR applies a second set of quality control on all ES cells to ensure that our customers get only cells that are best for GLT.
Production Centre QC
- 5′ LRPCR confirms correct integration of the allele on the 5′ side.
- 3′ LRPCR confirms correct integration of the allele on the 3′ side.
- TRPCR confirms presence of the 3′ loxP site.
EuMMCR Quality Control
The EuMMCR lab picks only the clones with the best QC results from the production lab for expansion. The quality control that EuMMCR applies contains the following tests:
1. Mycoplasma PCR: A PCR ensuring that the ES cell preparation is mycoplasma free. Every ES cell clone is tested for mycoplasma using the assay developed by Uphoff CC, Drexler HG., 2002. Any contaminated ES cell lines are removed from the resource.
2. loxP verification: The identity of each clone is confirmed by PCR comprising a gene specific primer (unique to each allele) and a primer immediately downstream of the 3′ loxP site (loxR). A band of the expected size indicates that the 3′ loxP site is present downstream of the "critical exon".
3. Genotyping: We develop a genotyping reaction using a short PCR strategy. This service provides the user with an easy to use genotyping reaction for ES cells and chimeric mice. The genotyping strategy can be one of the following:
Option 1: Two gene specific primers are designed upstream and downstream of the 3′loxP site (gene-for and gene-rev). During homologous recombination, the targeting cassette replaces endogenous DNA stretches. Option 1 genotyping only works well when the PCR fragment of the targeting cassette differs by ~50bp from the PCR fragment based on the wt allele. This size difference cannot be changed by design of alternative primers, it is a property of the mutated allele.
Option 2: Our favored strategy for developing this genotyping reaction. A gene specific primer in the 5′ homology arm (gene-5′arm) is designed and used with the LAR3 primer in the targeting cassette to detect the mutated allele. Another gene specific primer is designed in the 3′ homology arm (gene-3′arm) in order to detect the wt allele with a PCR fragment between the two gene specific primers.
Option 3: The mutated allele can be also be distinguished from the wt allele by performing a PCR with the gene-for and gene-rev primer (from Option 1) and digesting the resulting PCR fragment with SacI. The wt allele usually does not contain a SacI site, while there is a SacI site within the loxP sequence.
One of the three options (Option 2, Option1 or Option 3) digest will be performed and gel pictures and brief protocols will be delivered in a pdf document via email after the shipment of the cells.
4.Integration of the targeting cassette: To verify the correct integration of the targeting cassette into the wild type allele, we developed two different approaches.
Long Range PCR: We only send out clones that pass our 5′ and 3′ assays. We do not rely on the assay performed by the EUCOMM production lab but will perform this assay independently using the ES cell preparation as a template.
The EUCOMM quality control project has determined that only 82% of clones marked "EUCOMM ES cell clones with conditional potential" will pass a Southern blot assay independent of their PCR-based QC status. EuMMCR suspects that the remaining 18% are either mixed clones or irregular recombination events.
In case of "EUCOMM ES cell clones with conditional potential" with LRPCR′s verified on both the 5′ and the 3′ site the value for probability of passing a Southern blot a rises to 0.96.
Loss of Allele Assay: As an alternative method to verify the integration of the targeting cassette in the genome we use the so called "loss of allele"- assay (LoA-assay), which quantifies the number of copies of the native locus to which the mutation was directed. This is a qPCR based assay with a probe that binds to a wt-sequence, which is deleted when the "cassette" of the targeting vector is integrated. As a result the probe binds either twice, if both alleles are wt or only once if the ES cell clone is correctly targeted.
5. Chromosome Check and Copy Number: This is a high-throughput real time quantitative PCR method to easily check the quality of the homologous recombination and also the chromosomes of the ES cells. The copy number downstream loxP site is checked again, together with the copy number of present lacZ and neo sites in the genome. Further, the copy number of specified sequences on the chromosomes 8, 11 and Y are tested by this PCR test.
6. (optional) Chromosome Counting: During tissue culture and freezing processes, changes to the set of chromosomes (e.g. trisomy) can occur. The chromosome counting service is intended to ensure that users receive ES cells of which at least 50% have a normal set of chromosomes. The coromosome number is counted in 30 metaphase cells and users will only receive clones where more than 15 cells displayed a normal set of chromosomes.
Please note: Even though all of our ES cell clones passed this thorough quality control we still highly recommend to re-confirm the ES cell clone genotype by Southern blot analysis before blastocyst injection.
As a highly experimental resource there is NO WARRANTY or GUARANTY that the offered clones and vectors are suitable for any purpose. You may receive germline transmission from our clones although this might also depend on the targeted region, but you may not expect to do so if you order only one clone.
EUCOMM has determined the germ line transmission (GLT) rate of the resource to be roughly 60%. Therefore EuMMCR highly recommends users request 3 ES cell clones per gene to ensure GLT.
What quality control procedures are implemented by EUCOMM/the EuMMCR unit to ensure non contaminated ES cell clones?
The EUCOMM production labs have MAP testing performed with wt ES cell stocks used in electroporations in order to generate ES cell clones. Each individual knockout clone is not MAP tested.
The EuMMCR unit lab tests every ES cell clone that is expanded in the lab for mycoplasma infection. So far not a single ES cell clone has been mycoplasma positive.
As a quality control effort EUCOMM is producing 320 mouse lines in five mouse clinics in Europe. One of these mouse clinics, which is currently receiving roughly 10% of the EuMMCR unit's ES cell production, has all imported ES cell clones MAP tested.
We view this quality control effort as batch testing of our ES cell production.
EUCOMM and the EuMMCR unit are committed to a very high standard of controls. However, EUCOMM and the EuMMCR unit cannot offer a "sanitary certificate" or a guaranty of any kind, since it is in the nature of the resource that the EuMMCR unit ES cells are handled in laboratories outside our supervision.
Are EUCOMM ES cells male or female?
EUCOMM ES cells are male.
Which wt ES lines have been used to generate conditional knockout clones?
|wt ES cell line||background||Remarks|
|E14||129P2||Used for gene trap resource only. Widely used and well established. Suitable for injections on C57BL/6 blastocysts.|
|JM8.parental||C57BL/6N||GLT rate determined at the WTSI for JM8.parental ES cell clones: 62%|
|JM8.F6||C57BL/6N||JM8 subclone isolated at the WTSI. The "F" stands for "feeder dependent". Suitable for injections in BALB/c or C57BL/6J-Tyrc/c blastocysts. GLT rate determined at the WTSI for JM8.F6 ES cell clones: 62%|
|JM8.N4||C57BL/6N||JM8 subclone isolated at the WTSI. The "N" stands for "no feeders". Suitable for injections in BALB/c or C57BL/6J-Tyrc/c blastocysts. GLT rate determined at the WTSI for JM8.N4 ES cell clones: 69%|
|JM8A1.N3||C57BL/6N||JM8.F6 derived ES cell line by removing the retroposon element in the Agouti gene by gene targeting. These cells are heterozygous for Agouti (A/a). This provides a convenient dominant coat color marker to detect germline transmission in testcrosses to C57BL/6N mice. JM8A1.N3 cells are feeder independent. With a (so far) small number the GLT rate of JM8A1.N3 clones determined: 75%|
Is the Ordering of wt ES cells used in the EUCOMM program possible?
The ES cell lines JM8.F6, JM8.N4, and JM8A1.N3 are available from EUCOMM. If you want to order wt ES cells, please click here.
Are there any warnings and recommendations?
- Southern blot: The EUCOMM resource is a high-throughput gene targeting effort and for practical reasons, all clones have been characterised by our well-established quality control tests.
In a random sampling of the resource, Southern blot analysis with internal probes revealed complex events in approximately 15% of the ES cell clones. As such we recommend that customers order at least 3 different ES cell clones per gene, and use Southern blot analysis to verify correct targeting for all clones.
- Removal of floxed region (when used as complete knockout (targeted trap)): EUCOMM strongly recommends the removal of the floxed region prior to phenotypic analysis (especially for promoter-containing targeting cassettes as the promoter-driven neo is also removed) as this will ensure a true null allele.
- Germline transmission: The clonal germline transmission rate of EUCOMM targeted ES cells obtained by EUCOMM mouse production centers is approximately 50%. To maximize the chance of achieving germline transmission, EUCOMM strongly recommends microinjections of three independent ES cell clones. Optimal results are obtained from injections of C57BL/6 host blastocysts.