Removal of loxPFlanked Short Genomic Segment In Situ by Crossing Germline Transmitter Chimeras with a Stable Cre Transgenic Line

This approach requires the availability of a Cre-expressing transgenic line. In our laboratory, we have established a Cre recombinase-expressing transgenic mouse line (tgCre-1) by pronuclear injection of the hCMV Cre gene. Briefly, the insert purified from pBS185 plasmid (34) was directly injected at a concentration of 5 ng/mL into the pronucleus of zygotes fertilized by germline-transmit-ting males. This line is now routinely used to remove the loxP-flanked genomic piece efficiently from a targeted locus, by crossing this line with the targeted mouse (13a). This and other lines with similar properties (41) can be found in the Cre transgenic database mentioned previously (Subheading 1.1.4.4.).

1. Males homozygous or heterozygous for the loxP-flanked DNA sequence are crossed with Cre transgenic females.

2. Offspring are screened for the required recombination event by PCR (from ear punch-derived sample DNA) or Southern analysis (from tail biopsy sample-derived DNA).

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