Targeted Disruption of the PBR Gene in Steroidogenic Cells

In order to investigate further the role of PBR in steroidogenesis, we developed a molecular approach based on the disruption of PBR gene in the constitutive steroid producing R2C rat Leydig cell line by homologous recombination (61b). On the basis of the known rat PBR gene sequence, we designed two sets of primers that allowed us to amplify two fragments of the PBR gene from R2C cells genomic DNA by PCR. These PBR genomic DNA fragments were cloned and used to design the targeting construct. The targeting vector was constructed by positioning (i) the neo gene, conferring the neomycin resistance that allows for a positive selection of cells that have undergone homologous recombination, in between the two PBR genomic DNA fragments; and (ii) the Herpes Simplex Virus-tyrosine kinase gene, for the negative selection against cells that have randomly integrated the targeting construct, at the 3'-end of the second PBR genomic DNA fragment. The targeting vector was then transfected in R2C cells and selection was performed with G418 and ganciclovir (62). Four G418/Ganc-resistant cell lines were generated. PBR expression, examined by ligand binding, was absent in all four cell lines. In addition, the PBR-negative R2C cells produced minimal amounts (10%) of steroids compared with normal R2C cells. However, incubation with the hydrosoluble analog of cholesterol, 22R-hydroxycholesterol, increased the steroid production by the PBR-negative R2C cells, indicating that the cholesterol-transport mechanism was impaired. The genomic DNA characterization of the PBR-negative R2C cells is under investigation.

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