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Germ line transmission of loxP-flanked allele

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Hypomorphic ^ " Germ line allele? Conditional knockout knockout

Fig. 2. Strategy for generation of conditional knockout animals. When making a conditional knockout mouse strain it is possible to obtain a series of different alleles. The first possibility is that the insertion of loxP sites may result in a hypomorphic allele with reduced expression of the targeted gene. It is usually preferable to avoid creating a hypomorphic allele because this may produce a phe-notype in the homozygous form and thus prevent a clear interpretation of the observed phenotype in the tissue-specific knockouts. Mating of mice with the loxP-flanked allele to mice with ubiquitous expression of cre (deleter-ere mice) will result in a germ line deletion. Tissue-specific knockout mice are obtained by mating mice with the foxF-flanked allele with mice with tissue-specific expression of Cre recombinase.

Strategy for Tissue-Specific Knockout of Tfam

The cre-loxP recombination system of bacteriophage PI has been exploited to establish a conditional knockout system in the mouse that allows tissue-specific disruption of genes.8 This simple system is based on the insertion of short DNA sequences, loxP sequences, flanking the exons to be removed. The insertion of loxP sequences should ideally not affect the function of the gene. The two loxP sequences will recombine in the presence of Cre recombinase, thus deleting the intervening sequence.

The strategy we use for conditional knockout of Tfam is outlined in Figs. 2 and 3. There are two main considerations when constructing the targeting vector. The first is to ensure that the insertion of loxP sites and the neomycin resistance gene does not result in a hypomorphic allele, that is, a TfamloxP allele that has reduced Tfam protein expression. The second consideration is to ensure that cre-mediated disruption of the TfamloxP allele results in a null allele without residual Tfam protein expression.

8 H. Gu, J. D. Marth, P. C. Orban, H. Mossmann, and K. Rajewsky, Science 265, 103 (1994).

loxP k>xP

loxP k>xP

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