Selection Marker negative

Figure 19 Sample gel of the results of a PCR. Lane 1 is a 100-bp molecular marker lanes 2-6 are samples. The presence of the top bands (the species-specific endogenous gene) demonstrates that the PCR amplification was successfiil. Lack of the middle band (the introduced effect gene) and the bottom band (the selectable marker gene) in lane 3 indicates that sample is negative for the effect gene. Presence of all three bands in the remaining lanes indicates the samples are positive for the effect gene...

Fig. 3. Representation of (A) the two basic types of targeting vectors, (B) positive and negative selection markers, (C) the hit-and-run approach to introduce subtle mutations, and (D) the CRE/loxP recombinase system.

Homologous recombination occurs approximately 1000-fold less frequently than non-homologous recombination, therefore methods have been developed to enrich for homologous recombination events. A widely applied method includes the use of a positive and a negative selection marker and does not require the expression of the target gene in ES cells (Mansour et al., 1988). The targeting construct is based on a replacement-type vector... 

Herrero, M., de Lorenzo, V. Timmis, K. N. (1990). Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.Journal of Bacteriology, 172,6557-67. 

Targeting vector A vector containing a positive selection marker (e.g., neomycin-resistance gene), negative selection marker (thymidine kinase gene) and a multiple cloning site. 

Fig. 2. Mutagenesis strategy. A neo gene replaced one-third of the ORF and was used as a positive selectable marker. The HSV thymidine gene (HSV-ffc) was used for negative selection. The vector was linearized with Xbal digestion. The targeted events were screened by Southern blot. Two Xbal sites are in the outside of the targeting vector (the 3 -end one is just at the end of the vector, which cannot be digested by Xbal if the vector is randomly inserted). An outside probe is located at the 5 -end. The genotype of the mice can be easily identified based on the size of the hybridized bands, 6.7 kb (mutant) vs 6 kb (wild-type).

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. 

Clostridium spp. used for exemplification Wild type or mutant Negative selection marker Positive selection marker Selection criteria Plasmid example Principle genes inactivated/added References... 

Clostridium thermoceUum demonstrated one of the fastest growth rates on crystalline cellulose by the use of a highly efficient cellulosome [54]. However, the construction of stable transgenic strains has been hmited by the lack of effective genetic tools. Tripathi etal. [55] developed positive and negative selectable markers to allow for the selection of strains that undergo allele replacement. The deletion of the Idh and pta genes, and a 2000-h adaptation, led to a 4.2-fold increase in the ethanol yield compared to wild-type cells. 

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