Gene targeting creates an mHRA allele encoding a severely truncated protein in which of the coding sequence is deleted. Two correctly targeted clones were used for blastocyst injections.Targeted disruption of the mHRA gene by homologous recombination.Genomic organization and disruption strategy for mHRA depicting the gene, the targeting construct, and the targeted mHRA allele.Exons III to VI were replaced by the dominant selectable neomycinresistance marker transcribed in an antisense orientation.Polyclonal antibodies against the human HRA protein were used.These data suggest that mHRA and mHRB are functionally redundant for NER in vivo, extending our in vitro observations. Apparently, mHRA is not essential for mouse development, and mHRB can compensate for any additional functions of mHRA.This indicates that inactivation of mHRA aggravates the severe developmental defects caused by a mHRB deficiency UV survival curves of primary mHRA, mHRA, and mHRA E.Cells were exposed to different doses of UV. After d, the number of proliferating cells was estimated from the amount of radioactivity incorporated during a h pulse with thymidine.For each genotype, identical results were obtained with three other cell lines in primary mHRA, mHRA, and mHRA E.Incorporation of radioactivity was measured by autoradiography and grain counting. XPA fibroblasts were measured as a negative control.For each genotype, consistent results were obtained with three other independent cell lines after UV exposure of primary mHRA, mHRA, and mHRA E.After a h pulse labeling with uridine, cells were processed for autoradiography.MEF lines of wildtype, XPC, mHRAB, mHRAB, and mHRAB RNA synthesis recovery after UV irradiation of wildtype, XPC, and DKO E.Two independent experiments using two other DKO cell lines showed a similar effect on UDS and RNA synthesis recovery.Apparently, one out of four mHR copies is sufficient for normal NER activity.Thus, in the absence of both mouse RAD proteins, XPC is either hardly expressed at RNA or protein level, or unstable.Triton X, and subsequently immunolabeled with affinitypurified polyclonal antibodies against the human XPC protein. The UV sensitivity of DKO cells expressing hHRB was only partly rescued, perhaps because of humanmouse differences. Importantly, expression of hHRB induced an increase in the total amount of endogenous mouse mXPC, as shown by both immunoblot. Because the absence of mHR causes a strong reduction in mXPC, we reasoned that expression of exogenous XPC might bypass the repair defect of DKO cells.Cells were fixed by paraformaldehyde, followed by. Triton X permeabilization, and subsequently immunolabeled with affinitypurified polyclonal antihuman XPC. However, introduction of hXPCGFP appeared to restore endogenous mXPC levels as shown by immunoblot. Apparently, hXPCGFP has a transeffect on mXPC stability.To investigate the stabilizing effect of mHRB on XPC, we cotransfected hHRB with hXPCGFP cDNA into DKO cells.Stably transfected clones exhibited wildtype UV resistance. This is probably caused by a level of hXPCGFP expression still below the detection limit in most cells, because immunofluorescence using <a href="http://www.targetmol.com/compound/Kaempferol">sell
Kaempferol</a> antiHA monoclonals revealed that most cells expressed the tagged transgene. Cells with green nuclei also showed bright foci, corresponding with sites containing a local high DNA concentration visible after DAPI staining. These data indicate that the cotransfected hHRB acts as a stabilizing factor for both hXPCGFP and endogenous mXPC.