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GGTC German Gene Trap Consortium

 

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 Genotyping mice with a gene-trap mutation

 

 

(ref.: Floss T. and Wurst W. (2001): Functional genomics by gene-trapping in embryonic stem cells. In: Embryonic Stem Cell Models to Study Lineage Specific Differentiation in vitro: Methods and Protocols. Humana Press, Vol. 185: 347-380)

 

Identification of heterozygous mutants

Identification of homozygotes using quantitative hybridization

Identification of mutants using RFLP's

Identification of mutated loci by plasmid rescue

Identification of mutants by inverse PCR- generated probes

Identification of trapped loci by adapter-mediated PCR

Identification of homozygosity by breeding

 

 


Identification of heterozygous mutants

 

F1 generation mutants are easy to genotype by using probes or primers recognizing vector sequences. Dot blot analysis or a genomic PCR can be utilized in order to distinguish between positive and negative offspring and to give an estimate on copy number in the case of multiple integrations. In case the trapped gene is expressed in the tail tip, b gal staining of tail biopsies has been applied successfully.
Since some cells carry more than one integration, which are not necessarily in tandem, the mutant phenotype/lacZ staining pattern is not necessarily segregating with the genetrap vector in the F2 generation.

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Identification of homozygotes using quantitative hybridization


Internal probes or primers are not appropriate in order to distinguish between heterozygotes and homozygotes, unless an unrelated genomic probe is used as a loading control.

 

identification using quantitativ hybridization

 

In a quantitative hybridization experiment a vector and a non-vector related probe is used in order to estimate the copy number of the gene-trap vector integrations in the F2 generation. The copy number can be estimated both from a southern hybridization or from a simple dot blot. By using restriction enzymes which cut outside the vector, multiple integrations can be detected.

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Identification of mutants using RFLP's


Often, cDNA or EST fragments or PCR-products are available which can be used as external probes. In these cases, restriction fragment length polymorphisms (RFLP) can generally be found by utilizing restriction enzymes which cut within the gene-trap vector.

 

identification using RFLPs

 

RFLP's can be utilized in order to identify F2 gene-trap mutants.

In some reported cases the 5' or 3' RACE fragment has been successfully utilized as an external probe. However, RACE fragments are usually small and derived from distant exons which could make it difficult to find RFLP's. Since there is usually no information on the exon/intron structures of trapped genes the same problems apply to finding external primer pairs.

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Identification of mutated loci by plasmid rescue

 

Quantitative hybridization is subject to interpretation and therefore can not be the method of choice. Some researchers take advantage of the bacterial ori and amp to use a plasmid rescue in order to isolate flanking probes, which are outside to the integrated gene-trap vector. It is recommended to check for a retainment of bacterial sequences prior to plasmid rescue by PCR or dot blot methods. Araki et al. have reported the loss of the ampicillin resistence cassette in more than 60% of examined cases. Probably due to homologous recombination between the LTRs when utilizing retroviral trap-vectors only 20-30% of examined clones loose the bacterial sequences (Geoff Hicks, personal communication).

 

identification of mutated loci

 

Plasmid rescue as a means of isolating external probes from gene-trap mutations.
In order to perform plasmid rescue, enzymes need to be found, which will release genomic fragments of reasonable length. Genomic fragments over 20 kb have been cloned successfully. For transformation, it is recommended to use a bacterial strain with a defective DNA recombination system in order to be able to propagate repetitive sequences that are frequent in intronic DNA. A highly recommended strain is the stbl2 bacterial strain which are suitable for the cloning of unstable inserts such as retroviral sequences or direct repeats.

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Identification of mutants by inverse PCR- generated probes

 

In our hands, the most successful method by far in order to generate external genomic probes for yet uncharacterized loci is inverse PCR (Protocol: H.v. Melchner, pers. communication).

 

PCR generated probes

 

External probes for genotyping can be generated by RACE or inverse PCR.

 

Identification of trapped loci by adapter-mediated PCR

 

Recently, an adapter-mediated PCR approach in order to generate an external probe has been proposed. The protocol is straightforward in order to genotype mice with the scrambler mutation but can be adapted on genotyping gene-trap mutations.
Briefly, the protocol involves the digestion of genomic DNA, anchoring with a blunt-end adapter and subsequent PCR with both adapter-specific and gene-specific primers.

 

Identification of homozygosity by breeding

 

In case mutant mice are viable and fertile, a reliable way in order to verify homozygosity is achieved by breeding potential homozygotes to wildtype mice. In case of homozygosity naturally each of the offspring will be heterozygous for the trap mutation.

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