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

The long term goal of the Nuclear Receptor Signaling Atlas (NURSA) resides in unraveling the physiological and pathological functions of nuclear receptors (NRs) at the molecular, biochemical and cellular levels. This multi-oriented task requires complementary approaches in order to determine the specific function(s) and precise expression and receptor activity patterns for each individual conventional or orphan receptor. To attain this objective, we have chose to turn to technologies recently made available to engineer bacterial artificial chromosomes (BACs)

Expression and function profiling of orphan nuclear receptors using bacterial artificial chromosome (BAC) transgenesis.-1

<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>construct will place the expression of the reporter gene under the control of the promoter and regulatory regions present in the BAC and are likely to recapitulate the expression pattern of the NR considered. Note: it is also possible to generate knock-out constructs with this approach, providing that a eukaryotic promoter (Pr.) is introduced in the construct in order to allow selection of recombinant ES cells with an appropriate selection marker (Sm* – dual selection marker, for prokaryotic and eukaryotic selection). Homologous recombination allows for more complex designs than the one presented here. B. Activity trap setting. This particular design results in the replacement of the endogenous DNA-binding domain (DBD) by the Gal4 DBD. Activation of a reporter gene upon binding of the chimeric protein to Gal4 UAS sequences (inset) should parallel the activity of the endogenous NR. C. Ligand trap setting. A version slightly different from B. The expression of this fusion gene will activate reporter gene expression upon binding of LBD by (putative) ligand(s). A, B: regions of homology designed for recombination; Sm: Selection marker; Pr.: Eukaryotic promoter; DBD: DNA-Binding Domain; LBD: Ligand Binding Domain ; NR: Nuclear Receptor; frt: recombination sites for the Flt recombinase (for removal of the selection marker in a subsequent step)

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

<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