BioVector? NTCC? PL451-GFP大腸桿菌基因敲除熒光報(bào)告質(zhì)粒BioVector?載體

PL451 was constructed by introducing an FRT site upstream of Neo, and FRT and loxP sites downstream of Neo, in PGKNeobpA, a selection cassette that is commonly used for gene targeting in ES cells (Fig. ?(Fig.3A).3A). Similar to PL452, we introduced a bacterial EM7 promoter in between the PGK promoter and the coding sequence of Neo. This selection cassette works efficiently in both E. coli and mouse ES cells (data not shown). FRT is the DNA recognition site for Flp recombinase. DNA located between two FRT sites in mouse ES cells can be excised by transient expression of a genetically enhanced Flp recombinase (Flpe; Buchholz et al. 1998), which works well in ES cells. In this case, single FRT and single loxP sites were left behind at the targeted locus (Fig. ?(Fig.3A).3A). Only one Flpe recombination product is possible, which ensures that all excision products are the correct ones. Alternatively, the PL451 selection cassette can be removed after the conditional allele is introduced into the mouse germ line by breeding the mice to one of the mouse strains that expresses Flpe in the mouse germ line (Rodriguez et al. 2000). Subsequent expression of Cre recombinase will excise the entire DNA between the loxP sites located on either side of Evi9 exon 4, and create an Evi9 null allele. Cre can be expressed in the mouse germ line to create a germ-line null allele, or in somatic cells.

The PL451 selection cassette was introduced into the subcloned DNA in the same manner used to introduce the floxed Neo gene upstream of Evi9 exon 4. Evi9 exon 4, including both targeted regions, was sequenced to make sure that no undesired mutations were introduced during the recombination process. To functionally test the loxP and FRT sites in the targeting vector, the cko-targeting vector plasmid DNA was transformed into arabinose-induced EL350 and EL250 cells (EL250 cells have a Flpe gene under the control of the arabinose-inducible promoter, PBAD; Lee et al. 2001), respectively. Cells were plated on ampicillin plates to select for the plasmid. Plasmid DNA was prepared and digested to confirm the expected recombination patterns

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