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Adult ,8-Globin Gene (Homologous Recombination/Gene Targeting/Locus Control Region) W

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Adult ,8-Globin Gene (Homologous Recombination/Gene Targeting/Locus Control Region) W Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3177-3181, April 1993 Genetics Lethal thalassemia after insertional disruption of the mouse major adult ,8-globin gene (homologous recombination/gene targeting/locus control region) W. RONALD SHEHEE, PAULA OLIVER, AND OLIVER SMITHIES* Department of Pathology, University of North Carolina, 701 Brinkhous-Bullitt CB7525, Chapel Hill, NC 27599-7525 Contributed by Oliver Smithies, December 22, 1992 ABSTRACT Thalassemias are hereditary anemias caused ences in the two phenotypes can be explained based on by mutations that disturb the normal 1:1 balance of a- and competition of genes in the globin cluster for interaction with 3-globin chains that form hemoglobin. We have disrupted the the locus control region (LCR) (4-7) and for translational major adult 3-globin gene (bl) in mouse embryonic stem cells factors. by using homologous recombination to insert selectable se- quences into the gene. Mice homozygous for this insertional disruption of the bl gene (Hbb"l-2/HbbI-2) are severely anemic MATERIALS AND METHODS and die perinatally. In contrast, -60% ofmice homozygous for Cells and Electroporation. Gene targeting was done with deletion of the same gene (Hbbl*-1/Hbbl*-1) survive to adult- E14TG2a ES cells isolated from inbred strain 129 mice (8). hood and are much less anemic [Skow, L. C., Burkhart, B. A., The cells were cultured on embryonic fibroblast feeder layers Johnson, F. M., Popp, R. A., Goldberg, S. Z., Anderson, in Dulbecco's modified Eagle's medium (DMEM)/15% fetal W. F., Barnett, L. B. & Lewis, S. E. (1983) Cell 34, 1043- bovine serum/0.1 mM 2-mercaptoethanol/2 mM glutamine. 1052]. These different phenotypes have implications for the The targeting plasmid (see Fig. 1B) was introduced into the control of ,-globin gene expression. ES cells by electroporation by discharging a 200-IF capacitor charged to 300 V through a 5-mm-long chamber with a The a- and (3-globin polypeptide chains of hemoglobin are 100-mm2 cross-section. Cells (2 x 107) were used with a DNA encoded by multigene clusters that are generally conserved concentration of 5 nM. After electroporation, cells were throughout mammalian evolution. Within the clusters, the cultured at a density of either 5 x 106 or 2 x 106 on 100-mm- individual genes are regulated in both a temporal and tissue- diameter dishes and were exposed to 200 mg ofG418 (GIBCO) specific manner. There are five functional genes in the human per ml and 2 mM ganciclovir (Syntex, Palo Alto, CA). /3-globin gene cluster: E, an embryonic globin gene expressed Screening for Homologous Recombinants. One portion of primarily in yolk sac-derived cells from 3 to 8 weeks of each colony surviving exposure to the two drugs was sub- gestation; Gy and Ay, fetal globin genes expressed primarily cultured; another was pooled with five other colonies and in fetal liver-derived cells from 6 weeks ofgestation to -3 mo analyzed by PCR as described (9), except that 0.5 p.g ofDNA after birth; and 8 and 3, adult globin genes expressed pri- and 1 unit of Taq DNA polymerase were used in each marily in bone marrow-derived cells starting shortly before reaction. The PCR mixtures were amplified for 35 cycles with birth and persisting throughout adult life. The ,3 gene is 45 sec of denaturing at 92°C followed by annealing and responsible for 97-98% of adult 8-globin, and the 8 gene is extension at 65°C for 10 min. One primer, 5'-GCTAACCA- responsible for -2-3%. There are four functional genes in the GATTTGTGAGCTCAGGG-3', was specific for sequences mouse 3-globin gene cluster: bhl, an early embryonic globin in the target gene bl, and the other, 5'-TGGCGGACCGC- gene expressed primarily in yolk sac-derived cells from 9.5 to TATCAGGAC-3', was specific for sequences in the neo 12.5 days of gestation; y, a late embryonic globin gene After 20 of the reaction mixture was electro- expressed primarily in fetal liver-derived cells from 11.5 to gene. PCR, A1l 16.5 days of gestation; and bi (fImaJir) and b2 (fBminor), adult phoresed on an agarose gel, transferred to nylon membranes, globin genes first expressed at 9.5 days of gestation in yolk and hybridized to a 32P-labeled bl-specific probe (see Fig. sac-derived cells, then expressed in fetal liver-derived cells, 1C) designed to detect a 1.6-kb band expected when these then expressed in spleen, and finally expressed in bone two primers are used to amplify DNA from a correctly marrow-derived cells throughout adult life (1). The bl gene is targeted cell. Three pools gave a positive PCR signal. Indi- responsible for =80% ofadult /3-globin, and b2 is responsible vidual colonies from these pools were then tested by PCR, for -20% (2). The net synthesis of a- and 8-type globin and the positive colonies so identified were further analyzed polypeptide chains is normally balanced at 1:1. by Southern blots of digested genomic DNA. As a step toward generating mice unable to synthesize any Preparation of Blood. Blood from weanling mice, or day mouse adult f3globins, we have used homologous recombi- 18.5 embryos, was collected in heparinized hematocrit tubes. nation in embryonic stem (ES) cells to disrupt the mouse Blood smears made for some animals were stained in major adult 3-globin gene (bi) by inserting into its second Wright's stain. For other animals, the tubes were sealed and exon a bacterial gene (neo) that confers resistance to the drug centrifuged, and hematocrits were measured; the resulting G418. Mice homozygous for this insertional disruption ofthe packed red blood cells were then removed, washed once with bi gene die perinatally due to severe anemia. This phosphate-buffered saline, and lysed in cystamine-containing thalassemic phenotype is much more severe than expected buffer as described (10). Samples containing approximately because mice homozygous for deletion of the same gene can equal amounts of hemoglobin were electrophoresed on cel- survive (3). We propose a model suggesting how the differ- lulose acetate membranes (Helena Laboratories). The publication costs of this article were defrayed in part by page charge Abbreviations: LCR, locus control region; neo, neomycin; tk, thy- payment. This article must therefore be hereby marked "advertisement" midine kinase; ES, embryonic stem. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 3177 Downloaded by guest on October 1, 2021 3178 Genetics: Shehee et al. Proc. Natl. Acad. Sci. USA 90 (1993) RESULTS AND DISCUSSION EcoR I Bgl 11 Pst I Sph I S T ST S T S T Gene Targeting. The targeting plasmid and gene targeting scheme are illustrated in Fig. 1. The plasmid contains 6.2 kb *0 qp 20 of DNA, from a strain BALB/c mouse, that is homologous to the bi locus. A 1.1-kb fragment from pMClPOLA (Strat- agene) capable of conferring resistance to G418 is inserted at a BamHI site in exon 2 of the bl gene. The homologous segments were 0.8 kb on the 5' side and 5.4 kb on the 3' side 6.5 ofthe neo gene. Copies ofthe thymidine kinase (tk) gene from 5.4 0, herpes simplex virus 1 are inserted both 5' and 3' of the :. 4.7 homologous segments so that positive-negative selection could be used (11). After electroporation with the linearized targeting plasmid, 144 colonies were picked from a total of 604 that were resistant to both G418 and ganciclovir. PCR tests of these colonies indicated that three ofthe 144 colonies were correctly targeted. This number corresponds to a fre- quency of one targeted colony for every 1.5 x 106 starting I".n 2.2 cells. The selection with ganciclovir reduced the number of 4..4 surviving cells to about one-tenth of those surviving with 2.0 1.95 G418 alone. The three independently targeted cell lines identified by PCR were confirmed by Southern blots of digested genomic DNA. Fig. 2 shows a Southern blot ofDNA from the starting FIG. 2. Southern blots ofDNA from the starting and targeted cell cell line and from one ofthe targeted cell lines after digestion lines. The blots of DNA from the starting cell line E14TG2a (S) and with four restriction enzymes. The EcoRI digest shows an a targeted cell line (T) after digestion with fourenzymes. The location endogenous band of 7.3 kb (also present in DNA from the of the probe used is shown in Fig. 1. Sizes of bands are given in kb. starting cell line) and a band of 2.0 kb, which is the size expected from a correctly targeted chromosome. The Bgl II being mated to C57BL/6J females. The F1 offspring that are digest gives a 5.4-kb band (corresponding to the endogenous heterozygous for the disrupted bi gene (+/-) appear normal. locus) and the expected 6.5-kb band derived from a targeted In contrast, homozygous mutants (-/-) obtained after mat- locus. The Pst I digest shows a 2.2-kb endogenous band and ing heterozygotes are very pale and die either in utero close a 1.95-kb band expected for targeting. The Sph I pattern is to term or just after birth. Of five -/- mice that were alive also as expected: 20 kb for the endogenous locus and 4.7 kb at birth and were cared for by their mothers, none survived for the targeted locus. This and other blots were subsequently >4 hr. The phenotype of the mutant homozygotes is readily hybridized to a probe specific for the neo gene.
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