Mutant ES Cells
ES cells clones generated by gene targeting
All EUCOMM ES 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 analysed for correct targeting by PCR methods. Long range PCR (LRPCR) is applied to control fro 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 ES cell clones: a. "knockout first" and b. "targeted trap"
a. "Knockout first" alleles contain all components of the original targeting vector. They can be used for the generation of conditional alleles and lacZ-tagged null alleles.
b. "Targeted trap" alleles are missing the downstream loxP site. These alleles report gene expression by the lacZ-reporter and are highly likely to be function null mutations. However, they can not be modified into conditional alleles.
For each ES clone being sent out by the EuMMCR team, the presence of the downstream loxP site is confirmed by PCR. Furthermore, upon request we develop a PCR reaction enabling the user to genotype the ES cells as well as chimeric mice and their offspring. Our controls do presently not include Southern blot analysis. We highly recommend to users to re-confirm the ES cell clone genotype by Southern blot analysis before blastocyst injection.
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 succesful germline transmission will be indicated by black F1 offspring.
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.
Disclaimer
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.