Selection Cassette

Nrg1tm2Zhou Targeted mutation 2, Mingdong Zhou The majority of exon 13 and a portion of intron 13 were replaced with a neomycin selection cassette inserted by homologous recombination. The deleted region encoded the transmembrane domain... 

Fig. 4.1 Gene disruption by targeted mutation. To delete exon 2 (E2), a targeting construct was prepared with side arms containing homologous DNA upstream and downstream of exon 2, respectively. By recombination events in the upstream and downstream arms, exon 2 and part of the surrounding intronic region will be replaced by a neomycin selection cassette (neo), resulting in the inactivation of the gene.

Fig. 4.2 Detection of homologous recombinants by Southern blot. Restriction enzyme A cuts once in the homologous side arms of the targeting construct and once within the neomycin selection cassette. For Southern blot, the genomic DNA is digested with restriction enzyme A and hybridized with an external probe which stains bands of different size for homologous recombinants or the wild-type gene. In case of heterologous recombi...

If there is a need to insert in some distance of the selection cassette additional sequences (e.g., a loxP site or a point mutation), then an attempt should be made to introduce these sequences together with restriction sites that can be used for Southern blot analysis. Alternatively, genomic DNA of homologous recombinant clones can be checked by PCR for the presence of that sequence. 

As a selection cassette often disturbs the normal expression of a gene, it must be removed in conditional knock-out mice, and this can be achieved by introducing a floxed selection cassette. Homologously recombined ES cell clones are then transiently transfected in vitro with a cre recom-binase expression vector. If three loxP sites were to be introduced, different deletions would be possible (l- 2, 1-> 3, and... 

If the selection cassette also contains a negatively selectable marker such as tk, then cells that have lost the cassette can be selected for. The tk gene must be removed in vitro, as its expression impairs sperm motility in vivo and this results in sterile chimeric mice [21]. ES cells without a selection cassette can easily be retargeted by the original targeting construct to obtain homozygously recombined ES cells. 

As targeting vectors are normally in the range of 15 to 20 kb, cloning may become rather compheated. As a general strategy, we first try to subclone the two homologous arms, and then introduce selection cassette, loxP sites and other sequences by directed cloning with two sticky ends. 

The pMUW expression system [77] consists of two plasmids. Plasmid 1 carries the Ddp2 rep gene and a neo selection marker. Plasmid 2 does not contain a selection cassette, but has the Ddp2 origin of replication to keep the plasmid extrachromosomal it also contains a cassette for the expression of transgenes, consist-... 

Blaauw et al. [84] adapted a state-of-the-art Tet° system known from gene expression systems in mammalian cell fines (see Part IV, Chapter 1). Plasmid 1 (response plasmid) remains extrachromosomal, and has a hsr selection cassette and a tetracycline-responsive minimal promoter consisting of seven copies of the tetracycline operator sequence (tetO) upstream of an actl5 minimal promoter this is used... 

Place the neo selection cassette and transgene expression cassette onto the same plasmid. 

Fig. 3. Gene disruption using the Cre-/oJt approach. In the targeting vector, exon 3 is flanked by directly repeated lox sites with a positive-negative selection cassette within these sites. The ganciclovir-sensitive targeted cells can undergo Cre-mediated homologous recombination, resulting in excision of the selection vector and genomic sequences intervening the lox sites.

Ema, M., S. Takahashi, and J. Rossant. 2006. Deletion of the selection cassette, but not cis-acting elements, in targeted Flkl-lacZ allele reveals Flkl expression in multipotent mesodermal progenitors. Blood 107(1) 111-7. 

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