Section 36
Chapter 35,827

Targeted Gene Replacement of the Murine Alpha Globin Genes by Human Alpha Globin Results in High Level Expression

Ryan, T.M.; Pawlik, K.M.; Kapoor, P.; Divoky, V.; Townes, T.M.

Blood 100(11): Abstract No. 900


ISSN/ISBN: 0006-4971
Accession: 035826937

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Human alpha globin genes are not expressed in transgenic mice unless they are physically linked to, or coinjected with, locus control region (LCR) DNA sequences from either the human alpha or beta globin loci. This study examines the ability of the murine alpha globin locus to direct high level expression of human alpha globin after targeted gene replacement of the endogenous mouse alpha globin genes by human alpha globin. Positive-negative gene replacement constructs were designed to delete both of the adult mouse alpha globin genes, alpha1 and alpha2, and insert the human alpha1 globin gene alone or both the human alpha2 and alpha1 globin genes together. A 3.8 kb BglII/EcoRI DNA fragment of human alpha1 globin or an 8.4 kb SphI/EcoRI fragment with both human alpha2 and alpha1 globin was sandwiched between 4.0 kb of upstream and 6.4 kb of downstream mouse homology. A loxP flanked phosphoglycerate kinase (PGK) gene promoter driving the expression of a hygromycin (Hyg) selectable marker gene was inserted between the human alpha gene(s) and the downstream homology region. Nonhomologous recombinants were selected against by inclusion of a PGK driven HSV thymidine kinase gene inserted at one end of the constructs. Embryonic stem (ES) cell colonies were picked after one week of selection in hygromycin and ganciclovir. Southern blot and PCR analyses of DNA from these colonies were used to identify homologous recombinants. The efficiency of homologous recombination was 11% and 6% respectively for the alpha1 and alpha2/alpha1 constructs. Male chimeras were generated after blastocyst injection of three independent human alpha1 globin knockin (KI) ES cell lines. Chimeras from all three lines have transmitted the alpha1 KI modification through the germline. Mice heterozygous for the alpha1 KI are viable, fertile, and synthesize high levels of human alpha globin. HPLC analyses of the globin chain levels in hemolysates from heterozygous animals demonstrates that human alpha1 globin chains produced from this single KI gene are 38% of the total level of the two mouse alpha globin genes on the remaining wild type allele. These mice are the first demonstration of the endogenous murine alpha globin LCR enhancing the expression of human alpha globin. The human alpha1 KI mice have been bred to CMV-cre mice to remove the loxP flanked PGK-Hyg marker gene. Interestingly there was little change in the ratio of human alpha to mouse alpha globin after deletion of the marker. Additionally, mating two heterozygotes together produced homozygous human alpha1 KI mice that are also viable and fertile. These animals survive solely upon a human alpha/mouse beta hybrid hemoglobin (Halpha2Mbeta2). This KI line of adult human alpha globin mice will simplify the generation of future human hemoglobin mice by avoiding variable transgene copy number and random integration site effects; thereby, enabling new KI mouse models of hemoglobinopathies to be produced with precisely defined modifications more quickly and cheaply.

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