Correction of β Thalassemia by Homologous Recombination in Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 373-373 ◽  
Author(s):  
Thomas M. Ryan ◽  
Chiao-Wang Sun ◽  
Li-Chen Wu ◽  
Jin-Xiang Ren ◽  
Tim M. Townes
Keyword(s):  
Homologous Recombination ◽  
Es Cells ◽  
Globin Gene ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Microcytic Anemia ◽  
Globin Genes ◽  
Wild Type ◽  
Distribution Width ◽  
Erythrocyte Morphology

Abstract Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as β thalassemia and sickle cell disease. One genetic strategy for the correction of β thalassemia is to replace mutant or deleted β-globin alleles with a wild-type gene by homologous recombination in ES cells. Thalassemic mice that mimic the disorder have been generated by targeted gene deletion of the adult murine β-globin genes (PNAS 92: 9259–9263). We derived ES cells from our β-globin knockout mice and produced genetically identical mutant mice by injecting the ES cells into tetraploid embryos. These cloned β thalassemic mice have a severe microcytic anemia characterized by a marked reduction of the erythrocyte mean corpuscular volume (MCV), hemoglobin level (Hb), and hematocrit (Hct), and a marked increase in reticulocytes and red cell distribution width (RDW) compared to cloned wild-type control animals. In contrast to the normochromic, normocytic erythrocytes of wild-type clones, erythrocytes in peripheral blood smears of β thalassemic mice were hypochromic and exhibit extreme anisopoikilocytosis. A targeting construct containing 8.7 kb of mouse homology flanking a human γ- and β-globin gene cassette and a hygromycin marker gene was electroporated into the β thalassemic ES cells. After selection, DNA from 48 ES cell colonies was analyzed by PCR to identify homologous recombinants. Nineteen colonies (40%) had correctly integrated the human globin genes into the deleted mouse β-globin locus. Correctly targeted cells were injected into tetraploid blastocysts to produce mice that are derived solely from the corrected ES cells. These cloned mice synthesize high levels of human β-globin polypeptide that corrects the α- to β-globin chain imbalance, thereby eliminating the thalassemic erythrocyte morphology. The MCV, Hb, Hct, RDW, and reticulocyte levels in the blood of these mice are normal. These results demonstrate that a severe hemoglobinopathy can be cured after targeted gene replacement of a mutant gene(s) with a wild-type allele by homologous recombination in ES cells.

scholarly journals A Shortened Life Span of EKLF−/− Adult Erythrocytes, Due to a Deficiency of β-Globin Chains, Is Ameliorated by Human γ-Globin Chains

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1291-1299 ◽  
Author(s):  
Sai-Kiang Lim ◽  
James J. Bieker ◽  
Chyuan-Sheng Lin ◽  
Frank Costantini
Keyword(s):  
Life Span ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Es Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Mature Erythrocyte ◽  
Heinz Bodies ◽  
Globin Chains

Abstract Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF−/− ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF−/− colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of βh1-globin, which continued to be expressed at a very low level. The ratio of adult β-globin/α-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF−/− cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF−/− erythrocytes in adult animals must be short-lived, apparently due to the imbalance of β-versus α-globin chains, leading to the precipitation of excess α-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF−/− ES cells of a human LCR/γ-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

Long targeting arms do not increase the efficiency of homologous recombination in the β-globin locus of murine embryonic stem cells

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1531-1533 ◽  
Author(s):  
Zhi Hong Lu ◽  
Jason T. Books ◽  
Richard M. Kaufman ◽  
Timothy J. Ley
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Regulatory Sequences ◽  
Globin Genes ◽  
Gene Correction ◽  
Murine Embryonic Stem Cells ◽  
Alternative Approaches

Abstract The correction of mutant β-globin genes has long been a therapeutic goal for patients with β-thalassemia or hemoglobinopathies. The use of homologous recombination (HR) to achieve this goal is an attractive approach because it eliminates the need to include regulatory sequences in the therapeutic construct, and it eliminates mutagenesis induced by random integration. However, HR is a very inefficient process for gene correction, and its efficiency is probably locus dependent. The length of targeting arms is thought to be a determinant of targeting efficiency, so we compared the ability of standard (8-kb) versus very long (16-, 24-, and 110-kb) regions of homology to correct a mutant murine β-globin gene in embryonic stem cells. Increasing the length of the targeting sequences did not increase the efficiency of HR in this locus, suggesting that alternative approaches will be required to improve the efficiency of this approach for globin gene correction.

scholarly journals Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

1994 ◽  
Vol 14 (10) ◽  
pp. 6936-6943 ◽  
Author(s):  
P J Detloff ◽  
J Lewis ◽  
S W John ◽  
W R Shehee ◽  
R Langenbach ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Second Step ◽  
Globin Genes ◽  
Beta Globin ◽  
Es Cell ◽  
Beta Globin Gene ◽  
Targeting Strategy

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 beta-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 beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

scholarly journals Inhibited Neurogenesis in JNK1-Deficient Embryonic Stem Cells

2005 ◽  
Vol 25 (24) ◽  
pp. 10791-10802 ◽  
Author(s):  
Claudia R. Amura ◽  
Lindsay Marek ◽  
Robert A. Winn ◽  
Lynn E. Heasley
Keyword(s):  
Pc12 Cell ◽  
Neural Differentiation ◽  
Es Cells ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Signal Pathways ◽  
Es Cell ◽  
Wild Type ◽  
Base Pairs ◽  
Promoter Sequences

ABSTRACT The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.

scholarly journals Correction of sickle cell disease by homologous recombination in embryonic stem cells

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1183-1188 ◽  
Author(s):  
Li-Chen Wu ◽  
Chiao-Wang Sun ◽  
Thomas M. Ryan ◽  
Kevin M. Pawlik ◽  
Jinxiang Ren ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sickle Cell Disease ◽  
Homologous Recombination ◽  
Sickle Cell ◽  
Insertional Mutagenesis ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Cell Disease ◽  
Human Es Cells

Abstract Previous studies have demonstrated that sickle cell disease (SCD) can be corrected in mouse models by transduction of hematopoietic stem cells with lentiviral vectors containing antisickling globin genes followed by transplantation of these cells into syngeneic recipients. Although self-inactivating (SIN) lentiviral vectors with or without insulator elements should provide a safe and effective treatment in humans, some concerns about insertional mutagenesis persist. An ideal correction would involve replacement of the sickle globin gene (βS) with a normal copy of the gene (βA). We recently derived embryonic stem (ES) cells from a novel knock-in mouse model of SCD and tested a protocol for correcting the sickle mutation by homologous recombination. In this paper, we demonstrate the replacement of the human βS-globin gene with a human βA-globin gene and the derivation of mice from these cells. The animals produce high levels of normal human hemoglobin (HbA) and the pathology associated with SCD is corrected. Hematologic values are restored to normal levels and organ pathology is ameliorated. These experiments provide a foundation for similar studies in human ES cells derived from sickle cell patients. Although efficient methods for production of human ES cells by somatic nuclear transfer must be developed, the data in this paper demonstrate that sickle cell disease can be corrected without the risk of insertional mutagenesis.

Global ENU Mutagenesis Screen for Genetic Modifiers in Sickle Cell Disease Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3733-3733
Author(s):  
Thomas M. Ryan ◽  
Yongliang Huo ◽  
Sean McConnell
Keyword(s):  
Animal Model ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Positional Cloning ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Globin Genes ◽  
Genetic Modifiers ◽  
Cell Disease

Abstract Genetic modifiers of sickle cell disease (SCD) will be identified in an animal model of SCD after mutagenesis with the chemical mutagen N-ethyl-N-nitrosourea (ENU). This phenotype driven approach utilizes a third generation knock-in (KI) mouse model of SCD that reproduces most if not all of the pathology of the disorder. This model was produced by targeted gene replacement of the murine α-globin genes with human α-globin and the murine β globin genes with a human γ- and βs-globin gene cassette that mimics the fetal to adult hemoglobin switch that occurs in man. Sickle embryonic stem (ES) cells were derived from developing blastocysts isolated from female sickle mice that were mated with sickle males. Sickle ES cells were treated with ENU and a mutant library of independent subclones was established and archived. The optimal ENU dosage was empirically determined through a series of pilot experiments that measured the HPRT mutation frequency and the efficiency of producing sickle mice from the mutagenized cells by tetraploid embryo complementation. Animals harboring mutations that affect hematological indices, kidney function, or liver function are identified in the mutagenized sickle mice by comparison to cloned control sickle mice. Microsatellite linkage analyses of mutant offspring outcrossed to congenic SCD mice and direct sequence comparison to the murine genome will allow the positional cloning of modifier genes. Putative modifying factors will be positively confirmed by introducing the exact germline modification discovered during the ENU screen into the unmutagenized ES cells, followed by the direct examination of the phenotype in mice generated from the modified cells by cloning. These studies will define gene(s) responsible for the phenotypic variation in disease severity that is observed in the SCD population. By experimental design, the therapeutic benefit or detriment associated with each modifying gene(s) on the in vivo pathophysiology of sickle cell anemia will be tested directly in our animal model of this disorder.

Characterization of the LCR Dependent Activation of the Beta-Globin Genes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 377-377
Author(s):  
M. Bender ◽  
Tomoyuki Sawado ◽  
Tobias Ragoczy ◽  
Rachel Byron ◽  
Mark T. Groudine
Keyword(s):  
Homologous Recombination ◽  
Histone Methylation ◽  
Mutational Analysis ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Site Formation ◽  
Fish Analysis ◽  
Globin Genes ◽  
Pol Ii ◽  
Activated State

Abstract The activity of the β-globin locus is regulated by the locus control region (LCR) which in humans and mice is comprised of six DNaseI hypersensitive sites (HSs) located upstream of the β-like globin genes. Hispanic thalassemia, a naturally occurring deletion of the LCR plus 25kb upstream results in the failure to activate the β-globin locus at the levels of chromatin structure, transcription and replication. In order to examine how the HSs interact to regulate the endogenous β-globin locus, we have utilized homologous recombination for the mutational analysis of the endogenous murine β-globin LCR in embryonic stem cells, followed by the generation of mice. Previously we reported that deletion of the endogenous LCR by homologous recombination (ΔLCR) does not completely silence expression of the β-like genes, and has no measurable effect on nuclease sensitivity, promoter hypersensitive site formation or core histone hyperacetylation. Thus the LCR provides a necessary enhancer-like activity. In addition, while loss of the LCR leads to only a slight decrease in pre-initiation complex formation and Pol II binding to the promoter, there is a significant decrease in downstream polymerase and this correlates with a basal level of ser-5 phosphorylation of Pol II. To determine if the decrease in downstream Pol II observed along Δthe LCR allele is due to decreased release of polymerase from the promoter, or downstream polymerase pausing, KMnO4 in vivo foot-printing was done. Comparison of the 5′ end of WT and ΔLCR β-maj globin genes reveal similar patterns consistent with pol II pausing on both alleles, and suggesting that the LCR stimulates elongation by releasing promoter proximal paused polymerases. To further characterize LCR mediated activation and determine what characteristics vary with the level of transcription we have continued our analysis of mice with ΔLCR allele expressing at 1–4% of wild-type (WT), and mice with a deletion of HS 2 and 3 (Δ23) expressing at 30% of WT. The Δ23 allele was chosen as it demonstrates an intermediate transcriptional phenotype and is one of several double HS deletions that demonstrate that the LCR HSs contribute additively to globin gene transcription. To determine if histone modifications other than acetylation vary with LCR mutations and is associated with the level of transcription, the state of K4-tri and K79-di histone methylation was assayed along exon 3 of the β-maj gene of WT, Δ23 and ΔLCR alleles. While in some systems enrichment of methylation of K4 and K79 are associated with a permissive or activated state, we find increases in histone methylation with deletion of the LCR, and an intermediate degree of methylation with deletion of HS 2 and 3. Thus, surprisingly, increasing levels of transcription correlate with decreases in histone methylation. Finally, as enhancers increase the probability rather than the rate of expression we have analyzed WT, Δ23 and ΔLCR alleles with single allele transcription assays. RT-PCR reveals all Δ 23 and ΔLCR alleles express mRNA at 30 and 4% of WT respectively, consistent with bulk RNA analysis and suggesting that individual alleles are not permanently silenced. In contrast, primary transcript RNA FISH analysis demonstrates that the mutant alleles are less likely to be expressed, thus LCR HSs may affect the probability as well rate of transcription.

Gene targeting, principles, and practice in mammalian cells

1999 ◽  
Author(s):  
Paul Hasty ◽  
Alejandro Abuin
Keyword(s):  
Gene Targeting ◽  
Mammalian Cells ◽  
Es Cells ◽  
Globin Gene ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Fibroblast Cell ◽  
Chromosomal Locus ◽  
Target Locus

When a fragment of genomic DNA is introduced into a mammalian cell it can locate and recombine with the endogenous homologous sequences. This type of homologous recombination, known as gene targeting, is the subject of this chapter. Gene targeting has been widely used, particularly in mouse embryonic stem (ES) cells, to make a variety of mutations in many different loci so that the phenotypic consequences of specific genetic modifications can be assessed in the organism. The first experimental evidence for the occurrence of gene targeting in mammalian cells was made using a fibroblast cell line with a selectable artificial locus by Lin et al. (1), and was subsequently demonstrated to occur at the endogenous β-globin gene by Smithies et al. in erythroleukaemia cells (2). In general, the frequencies of gene targeting in mammalian cells are relatively low compared to yeast cells and this is probably related to, at least in part, a competing pathway: efficient integration of the transfected DNA into a random chromosomal site. The relative ratio of targeted to random integration events will determine the ease with which targeted clones are identified in a gene targeting experiment. This chapter details aspects of vector design which can determine the efficiency of recombination, the type of mutation that may be generated in the target locus, as well as the selection and screening strategies which can be used to identify clones of ES cells with the desired targeted modification. Since the most common experimental strategy is to ablate the function of a target gene (null allele) by introducing a selectable marker gene, we initially describe the vectors and the selection schemes which are helpful in the identification of recombinant clones (Sections 2-5). In Section 6, we describe the vectors and additional considerations for generating subtle mutations in a target locus devoid of any exogenous sequences. Finally, Section 7 is dedicated to the use of gene targeting as a method to express exogenous genes from specific endogenous regulatory elements in vivo, also known as ‘knock-in’ strategies. A targeting vector is designed to recombine with and mutate a specific chromosomal locus.

scholarly journals Repair of Double-Strand Breaks by Homologous Recombination in Mismatch Repair-Defective Mammalian Cells

2001 ◽  
Vol 21 (8) ◽  
pp. 2671-2682 ◽  
Author(s):  
Beth Elliott ◽  
Maria Jasin
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Strand Breaks ◽  
Mutant Cells

ABSTRACT Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93–101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2 −/− cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.

scholarly journals A Shortened Life Span of EKLF−/− Adult Erythrocytes, Due to a Deficiency of β-Globin Chains, Is Ameliorated by Human γ-Globin Chains

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1291-1299 ◽  
Author(s):  
Sai-Kiang Lim ◽  
James J. Bieker ◽  
Chyuan-Sheng Lin ◽  
Frank Costantini
Keyword(s):  
Life Span ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Es Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Mature Erythrocyte ◽  
Heinz Bodies ◽  
Globin Chains

Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF−/− ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF−/− colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of βh1-globin, which continued to be expressed at a very low level. The ratio of adult β-globin/α-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF−/− cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF−/− erythrocytes in adult animals must be short-lived, apparently due to the imbalance of β-versus α-globin chains, leading to the precipitation of excess α-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF−/− ES cells of a human LCR/γ-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

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