scription or translation. This can be accomplished either by inserting the cassette in a critical region or by replacing a portion of the endogenous gene with the selection cassette producing a deletion. The selection cassette usually has its own promoter and polyadenylation sequences. Note that such a mutation cannot properly be called a knockout, or assumed to be a null mutation, until evidence has been obtained that no gene product is produced in the mutant. Expression of a partial gene product might result in a phenotype other than the null phenotype.

Expression Reporting. A variation on a simple knockout construct can provide information on the expression pattern of the targeted gene. For this purpose, a reporter gene (e.g., the E. coli gene lacZ or the jellyfish gene for GFP) is used without a promoter. The targeting construct is made in such a way that the reporter gene is in frame and is driven off the targeted gene’s promoter following homologous recombination. In this way, the reporter-gene product will be produced in all cells that would normally express the targeted gene. Usually, the reporter gene is studied in a heterozygote so that the other allele provides a functional gene product.

Point Mutations. Various schemes have been devised to produce mutations that are more subtle than complete loss of function. These usually involve a targeting construct that replaces the endogenous sequence with a homologous sequence containing a point (or other type) mutation. The trick is then to remove the selectable marker. This is most commonly done by using the bacterial Cre recombinase system, which is similar to the FLP recombinase system described in Figure 7-4. In the targeting construct, the selectable marker is made to be flanked by lox-P sites, short DNA sequences that are the substrate for Cre recombinase. Correctly targeted cell clones are then transiently transfected with the Cre gene, whose encoded product causes recombination between the lox-P sites, popping out the intervening DNA. The targeted allele is then left with a point mutation and a single lox-P site, which, if located in an intron, has no effect on the targeted gene.

Conditional Mutations. The Cre recombinase system (similar to the FLP recombinase system) has proved to be extremely versatile in gene-targeting schemes. The greatest potential is perhaps in the production of so-called conditional and tissue-specific mutations. The targeting construct is similar to that described above for making point mutations. The difference, however, is that a third lox-P site is introduced into a neutral position in the endogenous gene, in addition to the lox-P-flanked selectable marker. Then, following transient Cre expression, some cells will be recovered in which the lox-P-flanked selectable marker has been removed but two lox-P sites still remain. Any further expression of Cre will result in the removal of the intervening DNA, producing a deletion that can be planned to result in a null mutation. Provided the construct has been engineered so that the two remaining lox-P sites do not interfere with endogenous gene expression, normal mice can then be made with this ES cell line following



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