Dr. Bouhassira has developed Recombinase-Mediated Cassette Exchange (RMCE), a CRE recombinase-based method that allows highly efficient site-specific chromosomal integration in mammalian cells including ES cells. RMCE is ideally suited to study gene regulation and epigenetic phenomena because one can identify integration sites that are subject to strong position-effects and then revisit these sites with modified constructs designed to avoid the position-effects. Eliminating position-effects will permit the rational engineering of artificial genetic loci (AGL) that the applicant envisions as cassettes containing arrays of genes plus regulatory elements sufficient to autonomously control transcription, chromatin structure and replication timing at random integration sites. Transgenes making up an AGL would then be expressed in a tissue and developmental stage-specific manner at levels that are totally predictable. The applicant has observed that mice with deletion of some of their linker histone genes were not subject to the age-dependent silencing of globin transgenes that occurs in normal mice.
Aim 1 is to determine which of the seven histone H1 variants are important for this particular type of position-effect. The applicant has observed strong position-effects at three tagged loci in MEL cells. Importantly, the cause of these position-effects appears to be local because at all three loci, the level or stability of expression depended primarily on the orientation of the cassette on the chromosome.
In Aim 2, the applicant proposes to biochemically characterize these position-effects by performing DNasel sensitivity and methylation studies, and to determine the cause of the orientation-dependence of position-effects.
In Aim 3, the applicant proposes to prevent position-effects by controlling the replication timing and site of initiation of replication of expression cassettes using either RMCE or homologous recombination.
In Aim 4, the applicant proposes to systemically investigate the interactions between 2 genes and their cis-regulatory elements placed at various chromosomal sites. This will help understand how multiple genes in a given locus influence each others expression and how these influences can be modified. Understanding how to regulate AGL will be useful for gene therapy, for drug production and for the creation of animal models of human diseases.
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