Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis.-0

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis."</p><p>Nuclear Receptor Signaling 2003;1():-.</p><p>Published online 16 Jun 2003</p><p>PMCID:PMC1402220.</p><p>Copyright © 2003, Nemoz-Gailliard et al. This is an open-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. </p>on construct is flanked by two homologous regions (A and B) for the targeted BAC clone and contains a prokaryotic selection marker (Sm) for the selection of recombinant-containing bacteria. Electrocompetent cells cells containing the BAC (BAC or PAC) are prepared and induced to express the recombination function (rec+). The recombinant construct is then introduced in these cells by electroporation and the recombinants are selected against the selection marker (Sm). B. Modification of a BAC plasmid by homologous recombination in the bacteria: circular recombination. The targeting plasmid consists of a selectable and counter-selectable marker (Sm–Csm), a temperature-sensitive origin of replication (ts-ori), the recA gene and two homology regions (A and B). In the first round of homologous recombination, recombination can take place through either homology arm (A or B; for simplicity, only recombination through A is shown here), which results in the formation of a co-integrant. The co-integrant is identified by selection for the selectable gene at a temperature that is not permissive for the ts-ori. In the second round of homologous recombination, resolution of the cointegrant is driven by applying counter-selection against the counter-selectable gene at a temperature that is permissive for the ts-ori. Resolution can either revert the co-integrant to the original target molecule or generate the recombinant