Mutant ES Cells
ES cell clones generated by gene targeting
All EUCOMM ES cell clones generated by gene targeting have been generated in a mouse ES cell line of the genetic background C57BL/6N. Breeding of EUCOMM mutant mice with C57BL/6N mice allows users to perform phenotyping studies in a completely isogenic context from F1 generation on.
ES cell clones generated by gene targeting in the EUCOMM program have been analyzed for correct targeting by PCR methods. Long range PCR (LRPCR) is applied to control for homologous recombination in the 5′ and 3′ homology arms. Another quality control PCR checks for the presence of the downstream loxP site, which is sometimes lost due to recombination events in the homology region between targeting cassette and critical exon.
In the EUCOMM database the user can find two different quality control levels of Knockout-first ES cell clones: a. "with conditional potential" and b. "without conditional potential (targeted trap)".
- Knockout-first Alleles (with Conditional Potential) contain all components of the original targeting vector. They can be used for the generation of conditional alleles and lacZ-tagged null alleles.
- Knockout-first Alleles (without Conditional Potential) are missing the downstream loxP site. These alleles report gene expression by the lacZ-reporter and can function as null mutations. However, they cannot be modified into conditional alleles.
For each ES cell clone being sent out by the EuMMCR team, the presence of the downstream loxP site is confirmed by PCR. In addition the EuMMCR genotyping team verifies the 5′ and 3′ LRPCR′s performed in the production genotyping. Furthermore, we develop a PCR reaction enabling the user to genotype the ES cells as well as chimeric mice and their offspring. Via qPCR we check for the number of integrations of the targeting cassette (neo and lacZ) and for chromosomal integrity by checking a gene of the mouse chromosomes 8, 11, and Y.
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.
For more detailed information about our quality controls, please click here.
As part of the EUCOMMTOOLS program a library of highly germline-transmissible C57BL/6N mouse embryonic stem (ES) cell clones harboring targeted deletions for 392 miRNA genes have been generated. For most of these targeted clones, chimerism and germline transmission can be scored through a coat color marker. The targeted alleles have been designed to be adaptable research tools that can be efficiently altered by recombinase-mediated cassette exchange to create reporter, conditional and other allelic variants. This miRNA knockout (mirKO) resource can be searched electronically (www.mousephenotype.org) and is available from EuMMCR. (Posser HM et al., 2011)
Cre Driver ES cell lines
In addition to the gene targeting knockout ES cells, Cre Driver ES cell lines have been generated in the EUCOMMTOOLS program. By crossing with EUCOMM/EUCOMMTOOLS knockout mouse lines, the Cre Driver lines allow users to create conditional mutations that are activated in distinct cell types, tissues or time points, or mutations that are activated through administration of a drug.
Most of the Cre Driver ES cell lines are generated by the EGFP-CreERT2 Knock-in strategy.
ES cell clones generated by gene trapping
The EuMMCR distributes the resource of EUCOMM gene traps generated in E14 mouse ES cells. The genetic background of E14 cells is 129P2.
The key feature of the gene trap vector used in EUCOMM is the FlipROSAβGeo cassette (see figure below). The EUCOMM gene trap vector is delivered to ES cells as a MMLV retrovirus. The promoterless trap cassette consists of a splice acceptor, a β–gal reporter, a neomycin resistance gene, and a polyadenylation signal. To achieve conditionality of the trap alleles, the gene trap vector utilizes the F1Ex technology developed by F.Schnütgen. Two pairs of heterotypic recombination sites for the Cre and FLP recombinases, respectively, are positioned in inverted orientation around the trap cassette. This allows inverting the cassette two times. The first inversion silences the mutation and creates a conditional allele. The second inversion reinstates the mutation.
More detailed information about the vector can be found under Protocols.
ES cell handling
Aliquots of ES cell clones are shipped in cryotubes on dry ice to customers and should be stored for long term storage in liquid nitrogen tanks. The procedures for the cell culture handling of the ES cells can be found under Protocols.
EUCOMM ES cell clones can be used to generate conditional knock-out mice via blastocyst injection or morula aggregation.
Targeted ES cell clones that have been generated in the C57BL/6N cell lines JM8.F6 or JM8.N4 carry the genotype a/a (Agouti null) and should be injected into blastocysts of BALB/c donor mice. This will give rise to black/agouti/white chimeras. These chimeras should be bred with C57BL/6N animals, and successful germline transmission will be indicated by black F1 offspring. (Pettitt SJ et al., 2009)
Targeted ES cell clones that have been generated in the C57BL/6N cell line JM8A1.N3 carry the genotype A/a (Agouti heterozygous) and can be injected into blastocysts of C57BL/6 or BALB/c donor mice. Depending on the breeding scheme, chimeras will be agouti/black or agouti/white, and germline transmission will be indicated after breeding with C57BL/6N animals by either agouti or black F1 offspring, respectively.
The E14 gene trap ES cells can be injected into blastocysts of C57BL/6 donors, and germ line transmission will be indicated after breeding with C57BL/6N animals by agouti F1 offspring.
The EUCOMM resource is a high-throughput gene targeting effort, and for practical reasons, all clones have been characterized by PCR only. In a random sampling of the resource, Southern blot analysis revealed complex events in approximately 15% of the ES cell clones. Thus, we highly recommend that individual investigators use Southern blot analysis to verify correct targeting of the gene for all clones obtained from the EUCOMM resource.