One of the long term goals of this laboratory is to develop genetically-based strategies for the treatment of sickle cell anemia and beta-thalassemia. The goal of this study is to determine whether homologous recombination can be developed as a strategy to repair mutant beta-globin genes in embryonic stem cells and primary hematopoietic progenitors. To accomplish these goals, we propose the following specific aims.
Specific Aim 1 : We will develop mice with a mutation similar to the betas globin mutation of humans by creating a beta6 mutation in the mouse beta-major globin gene using homologous recombination in embryonic stem cells. We will create embryonic stem cells lines that contain a mutation that causes an alanine yields isoleucine substitution in position 6 of the murine beta-major globin gene (beta6I), and that have a selectable marker cassette (PGK-neo) either retained or excised (via Cre-Lox mediated recombination) downstream from beta-major. These ES cells will be the starting material for Specific Aim 2, and will be used to make mice that bear the mutations. The hematopoietic cells of heterozygous mice with beta6I (and the excised PGK-neo cassette) form the starting material for Specific Aim 3.
Specific Aim 2 : We will define the efficiency of homologous recombination-mediated repair of the beta6 mutation in embryonic stem cells using targeting vectors of different sizes. To explore the relationship of targeting arm size and homologous recombination efficiency, we will create targeting vectors that contain a total of 8, 16, 60, or 110 kb of wild-type targeting DNA from the mouse beta-globin cluster, and compare the abilities of these vectors to correct the beta6I mutation in ES cells via homologous recombination.
Specific Aim 3 : We will determine whether hematopoietic progenitors have the ability to correct the beta6I mutation via homologous recombination, using the targeting vectors defined in Specific Aim 2. Functional targeting vectors defined in Specific Aim 2 will be used to determine whether hematopoietic progenitors and/or stem cells have the machinery to perform homologous recombination events within the beta-globin locus. Bone marrow cells purified from mice heterozygous for the beta6I mutation (and PGK-neo deleted) will be transfected with the targeting vectors using physical means of DNA delivery (i.e. electroporation or lipofection). These cells will be selected using either neomycin phosphotransferase or GFP expression (or both), and individual colonies derived from hematopoietic progenitors (LTC-IC and CFU-C) will be analyzed for the frequency of correction of the beta6I mutation using PCR-based techniques. These studies should allow us to determine whether homologous recombination can be rationally developed as a method for correcting mutations in the beta-globin locus within primary hematopoietic progenitor cells.
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