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Chapter 4 : Chromosome Manipulation by Cre-lox Recombination

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Abstract:

Spaital and temporal regulation of Cre activity has led to the development of conditional somatic mutagenesis strategies that allow more precise determination of gene function in metazoans and plants. These strategies have been rapidly and widely adopted, particularly in the study of gene-modified and transgenic mice. In addition, Cre’s modest biochemical requirements have led to the adoption of Cre for use in a variety of other DNA manipulation strategies both in vitro and in vivo. The molecular genetics of the yeast is extraordinarily well developed, particularly in comparison with other eukaryotes. The genetic approach to mapping the spatial organization of the genome in the nucleus promises to provide important insights into the role of chromosome disposition in DNA repair and gene expression. The first successful approach to achieving the on/off expression control needed for inducible recombination-mediated gene ablation in mice was the use of the interferon-inducible Mx promoter to drive expression. A second strategy for regulating expression in mice is based on the successful adaptations of the prokaryotic tetracycline repressor operator system to regulate gene expression in eukaryotes. Precise engineering of individual chromosomes themselves, with production of defined chromosomal inversions, deletions, and duplications, is now possible using Cre recombinase. Advances in the genetic manipulation of model organisms have consistently led to deeper appreciation and understanding of their basic biology and to insights into the etiology of human disease.

Citation: Sauer B. 2002. Chromosome Manipulation by Cre-lox Recombination, p 38-58. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch4

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Figures

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Figure 1

DNA recombination reactions catalyzed by Cre. Open arrows represent the 34-bp site, and thin horizontal arrows indicate the 13-bp inverted repeat elements of the site. Rearrangement of the arbitrary genetic markers A, B, C, and D show the consequences of Cre-mediated DNA excision, integration, and inversion. The sequence of the spacer or core region (ATGTATGC) of differs from that of the heterospecific site ( ) at only a single position (ATGTATAC).

Citation: Sauer B. 2002. Chromosome Manipulation by Cre-lox Recombination, p 38-58. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch4
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Image of Figure 2
Figure 2

Stabilization of plasmid partitioning by Cre recombinase. The P1 genome in is represented by a circle, the site by the small black box.

Citation: Sauer B. 2002. Chromosome Manipulation by Cre-lox Recombination, p 38-58. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch4
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Image of Figure 3
Figure 3

Conditional Cre-mediated gene activation. Mice carrying a STOP cassette (GenBank accession no. U51223) inserted between a promoter and a reporter or potentially lethal gene are mated with transgenic mice having expression under the control of promoters with the desired spatial-temporal pattern. Double transgenic progeny are thereby produced in which the STOP cassette has been removed in the desired spatial-temporal manner. Example transgenics use the adenovirus EIIa ( ), CMV or cytomegalovirus major immediate early ( ), and Figure 3 . α-CaMKII or α- calcium-calmodulin-dependent kinase II ( ) promoters.

Citation: Sauer B. 2002. Chromosome Manipulation by Cre-lox Recombination, p 38-58. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch4
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Image of Figure 4
Figure 4

Cre-mediated chromosomal targeting in ES cells. The thin lines represent the transfected plasmid DNA; thick lines represent chromosomal DNA sequences. The desired transgene is designated YFG (our avorite ene) and the plasmid replication origin (ori) is represented by the circle. (A) Mutated site strategy ( ). Mutation (a 4-bp deletion) of in one of the inverted repeat elements is represented by an asterisk. After intermolecular recombination, one of the product sites carries mutations in both arms or inverted repeat elements and is thus blocked from participating in Cre-mediated reversal of integration. (B) Double replacement recombination ( ). Different heterospecific sites having nonidentical spacer sequences are indicated by open () and filled () large arrows. Alternatively, two sites in inverted configuration have been used ( ). Double-crossover recombination between sites on the incoming plasmid and the resident chromosomal sites replaces the -bounded chromosomal interval with that on the plasmid. Integration can be selected by incorporation of a marker on the plasmid into the chromosome, or by negative selection for loss of a negative selectable marker from the chromosome.

Citation: Sauer B. 2002. Chromosome Manipulation by Cre-lox Recombination, p 38-58. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch4
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