Confirm targeting vectors to all Javelins and report status."

This sub-fragment of Neo provided the 3' arm of homology in the targeting vector.

Such genetic approaches rely on either linear or circular targeting vectors to carry out homologous recombination.

In step 3, a general targeting vector is constructed that will allow the introduction of transgenes to the Hnf3α/Neo locus.

Therefore, ES cells that randomly integrate this targeting vector would remain sensitive to G418.

To test this prediction we generated a targeting vector that contained the LacZ gene from E.coli.

The 5' arm of homology within the targeting vector defines where the transgene is positioned relative to the Hnf3 α locus.

The targeting vector was electroporated into ES cells, and 27 G418-resistant colonies were analyzed for each construct.

A detailed description of the pMP8 targeting vector, from which these sequences were derived, has been described previously [ 15 ] .

An appreciable amount of recombination was observed while trying to grow up the final targeting vector, resulting in an aberrant plasmid of smaller molecular weight.

It was subsequently confirmed to be the correct targeting vector by diagnostic PCR, restriction digests and sequencing (data not shown).

This is particularly true for tagged sequence mutagenesis, where one would like to predict by sequence alone the effects of the targeting vector on cellular gene expression.

The targeting vector also contains the HSV TK gene to allow for negative selection of non-recombinants.

In generating the Hnf3αLacZ targeting vector we deleted all genomic sequences lying 3' to the first intron of the Hnf3 α gene.

Firstly, successful patenting of enhanced targeting vector technology has provided a basis for commercialization of the cell-models which eventuate from the application of these technologies.

Transgene targeting vector (p3αΔNeo) The transgene-targeting vector used in these experiments was generated in multiple steps (Fig 2).

Once the locus is chosen a targeting vector is designed to introduce a pgk/loxP-Neo cassette that confers resistance to G418 in mammalian cells.

The process, designated "tagged sequence mutagenesis", involves sequencing short segments of DNA isolated from each mutation to identify genes disrupted by the targeting vector.

Therefore, the mixture of theses two plasmids was digested with KpnI which linearized both plasmids, and the correct targeting vector was gel purified.

This in turn implies that, following electroporation of this targeting vector into Hnf3αΔpgk-Neo ES cells, 100% of G418 resistant colonies would have undergone homologous recombination.

Materials and Methods Sources of Plasmids The components of the ROSA26 targeting vectors were a gift from Philippe Soriano.

To facilitate this we have designed a general targeting vector that can accommodate and allow expression of any open reading frame when targeted to the Hnf3 α locus in Hnf3αΔpgk-Neo ES cells.

In the approach described here, a targeting vector that contains exon 1 of the hprt gene along with a specific transgene is used to reconstitute Hprt activity in Hprt negative ES cells.

Targeting vectors that utilize homologous recombination are the tools or techniques that are used to knock-in or knock-out the desired disease causing mutation or SNP (single nucleotide polymorphism) to be studied.

Alternatively, the problem may be avoided by using other selectable markers, assuming their sequences lack cryptic splice sites or by using gene trap vectors that rely on splicing to activate the expression of genes carried by the targeting vector.