Replication allows inactivation of a knocked-in locus control region in inappropriate cell lineages

Abstract

To study the influence of a locus control region (LCR) on the expression of a highly characterized, developmentally regulated locus, we have targeted the hCD2-LCR as a single copy into the endogenous mouse CD8 gene complex. Two knock-in mouse lines that differ in the integration site of the hCD2-LCR within the mCD8 gene complex were generated, and the influence on expression of the CD8 coreceptor was assessed. In these mice the normal developmental silencing of the CD8 genes in the CD4 lineage is deregulated, and the mice develop CD4 + cells that also express the CD8 genes. This is accompanied by the physical maintenance of the CD8 genes within an extended loop away from their subchromosomal territory. Further analysis of these mice revealed unexpected fluid chromatin dynamics, whereby the LCR can be initially dominant over the endogenous CD8 gene-repressive regulatory processes present in CD4 + cells but is continuously contested by them, resulting in the eventual inactivation of the inserted LCR, probably as a result of multiple rounds of replication.CD8 locus | hCD2 locus control region | knock-in | lymphocyte development | chromatin structure

Modulation of the Murine CD8 Gene Complex Following the Targeted Integration of Human CD2-Locus Control Region Sequences

“…CD8/ lxLCRlx knock-in mice negative for a Cre transgene deregulate the expression of the CD8 coreceptor, as seen previously in knock-in mice with constitutive insertion of the hCD2-LCR (Fig. 1B-E) (22).…”

“…In CD8/LCR (cII-III) knock-in mice with constitutive insertion of the hCD2-LCR, a reduction in CD4 + (CD8 + ) cells with age has been detected. Characterization of those mice indicated a link between the attenuated influence of the hCD2-LCR on CD8 coreceptor and the proliferation history of a cell (22). However, in dLck-iCre + CD8/ lxLCRlx knock-in mice, the decrease in CD4 + (CD8 + ) cells is more pronounced as a result of the deletion of the LCR (Fig.…”

“…To generate the targeting constructs for insertion of DNaseI HS1,2 or HS3 of the hCD2-LCR, a similar approach was used to that previously described (22). A 1.3-kb HindIII-XmnI fragment (HS1,2) or a 0.8-kb XmnIHindIII fragment (HS3) of the LCR was ligated into a HindIII/EcoRV linearized pBluescript SK plasmid containing the neomycin gene flanked with loxP sites (23).…”

“…Neoresistant ES cell clones for each targeting event were subjected to Southern blot analysis and hybridized with probes outside their homology region (22). Clones that had undergone correct homologous recombination were expanded and analyzed further using additional probes to confirm the integrity of the targeted CD8 gene complex.…”

“…Southern blots were set up to immobilize the samples on Hybond XL nylon membranes (GE Healthcare) and hybridized with probes labeled with [ 32 P]dCTP (GE Healthcare). DNA probes specific for the CD8 locus were used to identify the targeted and wild-type or deleted CD8 allele (22).…”