ARTIFICIAL GENETIC LOCI--UNDERSTANDING POSITION-EFFECTS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date5/1/004/30/01

Funding

  • National Institute of Diabetes and Digestive and Kidney Diseases: $253,641.00

ASJC

  • Genetics
  • Molecular Medicine
  • Molecular Biology
  • Cell Biology
  • Developmental Biology

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