We have used a "plug and socket" targeting technique to generate a mouse model of 130-thalassemia

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT-embryonic stem (ES) cells close to the chosen locus, in this case the j8-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse 1-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult 0-globin genes. The other replaced the same region with the human I-globin gene containing the mutation responsible for sickle cell anemia.Gene targeting in embryonic stem (ES) cells has made possible the construction of mice with predetermined mutations, including null mutations in many genes associated with human disease. Many genetic disorders, however, are not due to null mutations. In fact, at many disease-associated loci, the diversity of mutations causes a similar diversity in symptoms.For example, different mutations in the P-globin locus cause congenital Heinz body hemolytic anemia, familial cyanosis, sickle cell anemia, and thalassemias varying in severity (25). Mutational diversity consequently poses a challenge in designing mouse models of human disease, since each mutation must be targeted to the same locus as a separate recombination event.Conventional gene targeting involves positively selecting cells that have incorporated exogenous DNA, which includes a selectable marker such as the neo gene linked to sequences homologous to the target locus, followed by screening these transformants for cells in which the desired homologous recombination event has occurred (21). For some recombination events, thousands of colonies may have to be screened to find those that are targeted (10, 19), although the number can often be reduced by adding a negatively selectable marker such as the herpes simplex virus (HSV) tk gene to the construct (14). Altering a gene in many different ways by these procedures requires repeating the selection and screening for each modification. Strategies have therefore been devised to eliminate the repeated screening (1, 22, 26). For example, Askew et al.(1) used a two-step method to repeatedly modify the cx2-Na,KATPase gene. In the first step, both the neo and HSV tk genes were inserted into the target locus by conventional gene targeting. In the second step, the target gene was modifie...

The βIVS-2-654 C→T mutation accounts for approximately 20% of β thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to β0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the “plug and socket” method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult β globin genes with a single copy of the human βIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse β globin chains and no human β globin, and have a moderate form of β thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.

The βIVS-2-654 C→T mutation accounts for approximately 20% of β thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to β0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the “plug and socket” method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult β globin genes with a single copy of the human βIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse β globin chains and no human β globin, and have a moderate form of β thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.

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