Abstract 1058 The zebrafish is an important animal model for stem cell biology, cancer, and immun... more Abstract 1058 The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines, particularly in the context of transplantation experiments that distinguish between autologous and allogeneic tissues. Major histocompatibility (MH) genes are considered the most polymorphic genes in vertebrates, yet this immense variation occurs while maintaining conserved roles in antigen presentation. Histocompatibility in zebrafish remains poorly understood, requiring the identification of the classical MH genes as well as an evaluation of the variation between haplotypes. Although at least 11 putative zebrafish Class I MH U lineage genes have been isolated from cDNA libraries, their genomic organization and haplotype assignments remain uncharacterized for the majority. We focused our study on a set of diverse zebrafish Class I MH genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching was previously shown to be associated with improved engraftment after transplantation. Inside the conserved psmb8 and tpsn flanking gene regions on chromosome 19, Class I MH haplotypes can differ markedly among zebrafish strains including Tubingen and AB. Interestingly, the distinct haplotypes at chromosome 19 appear to maintain non-overlapping sets of genes, and also have gene copy number differences. For example, haplotype A contains the genes mhc1uda, mhc1uea, and mhc1ufa, with tap2 genes located in between each set of MH genes. In contrast, haplotype B contains mhc1uba and mhc1uca genes that are separated instead by a tapbp gene. Among these two highly divergent haplotypes, mhc1uea and mhc1uda from haplotype A appear to be more closely related to one another than to either of the MH genes on haplotype B. Similarly, the mhc1uba and mhc1uca genes from haplotype B share higher levels of sequence identity than in comparison with genes from haplotype A. These findings differ markedly from the MH genes in mammals where the highest similarity is between alleles of the same Class I gene (for example, HLA-A in humans), instead of the other genes on the same haplotype (eg. HLA-B and HLA-C). In addition, the zebrafish MH gene sequences are very closely related to MH genes from other fish species. As an example, mhc1ufa is more similar to a Class I MH gene from gibuna carp than to other zebrafish MH genes. These data in zebrafish indicate that selected Class I MH gene polymorphisms have been preserved within the species since before the species diverged from other teleost fishes, suggesting a selective advantage for this unique form of MH diversity. To determine mRNA expression levels of the Class I MH genes, quantitative PCR was performed on liver, spleen, kidney marrow, testis, gill, intestine and heart from zebrafish that were homozygous for either haplotype A or haplotype B. Within haplotype A, expression of mhc1uda, mhc1uea and mhc1ufa is relatively equivalent, with mhc1ufa expressed at the highest levels overall. For haplotype B, mhc1uba has the highest levels in all tissues examined, while mhc1uca expression is 10–100 fold lower and somewhat variable between tissues. Putative peptide anchor residues are highly conserved between species. Those anchor residues that are conserved in all species including mammals, as well as two additional residues that are conserved in teleosts, are also all conserved in mhc1uda, mhc1uba, and mhc1uca. In contrast, mhc1uea and mhce1ufa each have single amino acid substitutions at critical residues, K146N and Y59F, respectively. These substitutions do not necessarily disqualify these molecules from consideration as classical MH Class I genes. In conclusion, we propose that mhc1uba and mhc1uda function as classical MH Class I genes within divergent haplotypes, based on mRNA expression levels and tissue distribution, as well as sequence properties including conservation of putative peptide anchor residues. Using these same criteria, additional zebrafish Class I MH genes may be identified to serve similar roles. These predictions require verification via additional functional transplantation experiments that are currently underway in our lab. Defining the functional zebrafish Class I MH genes will provide an important foundation for future studies in immunology and transplantation. Disclosures: No relevant conflicts of interest to declare.
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for 15% of ... more T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. While initial treatment of T-ALL has improved, relapse is common and is associated with a poor prognosis. As with other tumors, T-ALL is genetically heterogeneous and relapse is driven at least in part by a subpopulation of cells called leukemia initiating cells (LICs). Capable of regrowing the entire tumor from a single cell, failure to eliminate these LICs is hypothesized to be the major determinant of relapse. Therefore understanding the genetic mechanisms that drive LICs may lead to new therapeutic targets that are likely to enhance rates of cure. The data presented here indicate that histone deacetylase 1 (hdac1) is an important regulator of LICs in T-ALL. Histone deacetylases (hdacs) modify chromatin structure and regulate gene expression by removing acetyl groups from histones and other proteins. As hdacs are aberrantly expressed in hematopoietic malignancies, and hdac inhibitors are used to treat some cancers, we hypothesized that hdac1 might play a role in leukemogenesis. To explore this question, we generated T cell acute lymphoblastic leukemias by overexpressing the murine c-Myc oncogene and the fluorochrome mCherry under the rag2 promoter in hdac1 haploinsufficient zebrafish. Tumor incidence and latency were not significantly different for hdac1+/- and wild type (WT) tumors. Mean tumor latency was 42.3 days post fertilization (dpf) for hdac1+/- fish and 47.9 dpf for their WT siblings. Likewise overall survival was not different. Mean survival was 58.0 dpf for hdac1+/- fish, and 64.6 dpf for WT fish. In contrast, when primary leukemia cells were transplanted into syngeneic recipient fish, the tumor cells from hdac1 haploinsufficient fish grew at a slower rate when compared to tumor cells from WT fish. After transplanting 1x105 primary WT or hdac1+/- tumor cells intraperitoneally into syngeneic zebrafish recipients, the transplanted WT tumors grew more aggressively compared to the hdac1+/- tumors. By 21 days post transplant, 92% of the WT tumors (n=22 of 24) had spread from the site of injection into the thymus and other organs, while most of the hdac1+/- tumors were only growing at the site of injection. Only 8.3% (n=1 of 12) of the hdac1+/- tumors had spread past the local injection site at this time point. We found that the slower rate of growth was not due to differences in proliferation, as determined by EdU incorporation. Hdac1+/- tumors had a mean percentage of EdU incorporation of 6.0 ± 4.0% (n=12), and WT tumors had 6.4 ± 4.4% (n=7); (p=0.8). In contrast, limit dilution transplant assays showed a higher frequency of leukemia initiating cells in the WT tumors (1 in 44 cells) compared with the hdac1+/- tumors (1 in 135 cells)…
Genetic modifiers of sickle cell disease (SCD) will be identified in an animal model of SCD after... more 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.
Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout an... more Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide some level of immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby challenging our ability to understand the evolutionary origin and specific function of these genes. In order to better understand the diversity of NILT genes and their possible roles in immune function, we identified 5 NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3 Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternate haplotype for this same genomic region. We then identified transcripts that define 43 different NILT genes and reflect an unprecedented range of sequence diversity and combinatorial diversity of Ig do...
A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of th... more A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the ...
Proceedings of the National Academy of Sciences, 2016
Significance Antigen presentation genes are exceptionally polymorphic, enhancing immune defense. ... more Significance Antigen presentation genes are exceptionally polymorphic, enhancing immune defense. Polymorphism within additional components of the MHC pathway, particularly the antigen processing genes, may also shape immune responses. Using transcriptome, exome, and whole-genome sequencing to examine immune gene variation in zebrafish, we uncovered several antigen processing genes not found in the reference genome clustered within a deeply divergent haplotype of the core MHC locus. Our data provide evidence that these previously undescribed antigen processing genes retain ancient alternative sequence lineages, likely derived during the formation of the adaptive immune system, and represent the most divergent collection of antigen processing and presentation genes yet identified. These findings offer insights into the evolution of vertebrate adaptive immunity.
Cooley’s Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin ... more Cooley’s Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This study reports novel preclinical mouse models of CA that survive solely on human fetal hemoglobin at birth and are blood transfusion dependent for life upon completion of their human fetal to adult hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse αglobin genes with human α globin and the adult mouse β globin genes with a delayed switching human γβ° or γδβ° globin gene cassettes. The nonfunctional human β° globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous humanized CA mice express 100% human hemoglob...
Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin ge... more Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expres...
During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucl... more During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and β globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells pro...
A preclinical humanized mouse model of β thalassemia major or Cooley anemia (CA) was generated by... more A preclinical humanized mouse model of β thalassemia major or Cooley anemia (CA) was generated by targeted gene replacement of the mouse adult globin genes in embryonic stem cells. The mouse adult α and β globin genes were replaced with adult human α globin genes (α2α1) and a human fetal to adult hemoglobin (Hb)–switching cassette (γHPFHδβ0), respectively. Similar to human infants with CA, fully humanized mice survived postnatally by synthesizing predominantly human fetal Hb, HbF (α2γ2), with a small amount of human minor adult Hb, HbA2 (α2δ2). Completion of the human fetal to adult Hb switch after birth resulted in severe anemia marked by erythroid hyperplasia, ineffective erythropoiesis, hemolysis, and death. Similar to human patients, CA mice were rescued from lethal anemia by regular blood transfusion. Transfusion corrected the anemia and effectively suppressed the ineffective erythropoiesis, but led to iron overload. This preclinical humanized animal model of CA will be useful ...
Abstract 1058 The zebrafish is an important animal model for stem cell biology, cancer, and immun... more Abstract 1058 The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines, particularly in the context of transplantation experiments that distinguish between autologous and allogeneic tissues. Major histocompatibility (MH) genes are considered the most polymorphic genes in vertebrates, yet this immense variation occurs while maintaining conserved roles in antigen presentation. Histocompatibility in zebrafish remains poorly understood, requiring the identification of the classical MH genes as well as an evaluation of the variation between haplotypes. Although at least 11 putative zebrafish Class I MH U lineage genes have been isolated from cDNA libraries, their genomic organization and haplotype assignments remain uncharacterized for the majority. We focused our study on a set of diverse zebrafish Class I MH genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching was previously shown to be associated with improved engraftment after transplantation. Inside the conserved psmb8 and tpsn flanking gene regions on chromosome 19, Class I MH haplotypes can differ markedly among zebrafish strains including Tubingen and AB. Interestingly, the distinct haplotypes at chromosome 19 appear to maintain non-overlapping sets of genes, and also have gene copy number differences. For example, haplotype A contains the genes mhc1uda, mhc1uea, and mhc1ufa, with tap2 genes located in between each set of MH genes. In contrast, haplotype B contains mhc1uba and mhc1uca genes that are separated instead by a tapbp gene. Among these two highly divergent haplotypes, mhc1uea and mhc1uda from haplotype A appear to be more closely related to one another than to either of the MH genes on haplotype B. Similarly, the mhc1uba and mhc1uca genes from haplotype B share higher levels of sequence identity than in comparison with genes from haplotype A. These findings differ markedly from the MH genes in mammals where the highest similarity is between alleles of the same Class I gene (for example, HLA-A in humans), instead of the other genes on the same haplotype (eg. HLA-B and HLA-C). In addition, the zebrafish MH gene sequences are very closely related to MH genes from other fish species. As an example, mhc1ufa is more similar to a Class I MH gene from gibuna carp than to other zebrafish MH genes. These data in zebrafish indicate that selected Class I MH gene polymorphisms have been preserved within the species since before the species diverged from other teleost fishes, suggesting a selective advantage for this unique form of MH diversity. To determine mRNA expression levels of the Class I MH genes, quantitative PCR was performed on liver, spleen, kidney marrow, testis, gill, intestine and heart from zebrafish that were homozygous for either haplotype A or haplotype B. Within haplotype A, expression of mhc1uda, mhc1uea and mhc1ufa is relatively equivalent, with mhc1ufa expressed at the highest levels overall. For haplotype B, mhc1uba has the highest levels in all tissues examined, while mhc1uca expression is 10–100 fold lower and somewhat variable between tissues. Putative peptide anchor residues are highly conserved between species. Those anchor residues that are conserved in all species including mammals, as well as two additional residues that are conserved in teleosts, are also all conserved in mhc1uda, mhc1uba, and mhc1uca. In contrast, mhc1uea and mhce1ufa each have single amino acid substitutions at critical residues, K146N and Y59F, respectively. These substitutions do not necessarily disqualify these molecules from consideration as classical MH Class I genes. In conclusion, we propose that mhc1uba and mhc1uda function as classical MH Class I genes within divergent haplotypes, based on mRNA expression levels and tissue distribution, as well as sequence properties including conservation of putative peptide anchor residues. Using these same criteria, additional zebrafish Class I MH genes may be identified to serve similar roles. These predictions require verification via additional functional transplantation experiments that are currently underway in our lab. Defining the functional zebrafish Class I MH genes will provide an important foundation for future studies in immunology and transplantation. Disclosures: No relevant conflicts of interest to declare.
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for 15% of ... more T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. While initial treatment of T-ALL has improved, relapse is common and is associated with a poor prognosis. As with other tumors, T-ALL is genetically heterogeneous and relapse is driven at least in part by a subpopulation of cells called leukemia initiating cells (LICs). Capable of regrowing the entire tumor from a single cell, failure to eliminate these LICs is hypothesized to be the major determinant of relapse. Therefore understanding the genetic mechanisms that drive LICs may lead to new therapeutic targets that are likely to enhance rates of cure. The data presented here indicate that histone deacetylase 1 (hdac1) is an important regulator of LICs in T-ALL. Histone deacetylases (hdacs) modify chromatin structure and regulate gene expression by removing acetyl groups from histones and other proteins. As hdacs are aberrantly expressed in hematopoietic malignancies, and hdac inhibitors are used to treat some cancers, we hypothesized that hdac1 might play a role in leukemogenesis. To explore this question, we generated T cell acute lymphoblastic leukemias by overexpressing the murine c-Myc oncogene and the fluorochrome mCherry under the rag2 promoter in hdac1 haploinsufficient zebrafish. Tumor incidence and latency were not significantly different for hdac1+/- and wild type (WT) tumors. Mean tumor latency was 42.3 days post fertilization (dpf) for hdac1+/- fish and 47.9 dpf for their WT siblings. Likewise overall survival was not different. Mean survival was 58.0 dpf for hdac1+/- fish, and 64.6 dpf for WT fish. In contrast, when primary leukemia cells were transplanted into syngeneic recipient fish, the tumor cells from hdac1 haploinsufficient fish grew at a slower rate when compared to tumor cells from WT fish. After transplanting 1x105 primary WT or hdac1+/- tumor cells intraperitoneally into syngeneic zebrafish recipients, the transplanted WT tumors grew more aggressively compared to the hdac1+/- tumors. By 21 days post transplant, 92% of the WT tumors (n=22 of 24) had spread from the site of injection into the thymus and other organs, while most of the hdac1+/- tumors were only growing at the site of injection. Only 8.3% (n=1 of 12) of the hdac1+/- tumors had spread past the local injection site at this time point. We found that the slower rate of growth was not due to differences in proliferation, as determined by EdU incorporation. Hdac1+/- tumors had a mean percentage of EdU incorporation of 6.0 ± 4.0% (n=12), and WT tumors had 6.4 ± 4.4% (n=7); (p=0.8). In contrast, limit dilution transplant assays showed a higher frequency of leukemia initiating cells in the WT tumors (1 in 44 cells) compared with the hdac1+/- tumors (1 in 135 cells)…
Genetic modifiers of sickle cell disease (SCD) will be identified in an animal model of SCD after... more 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.
Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout an... more Multiple novel immunoglobulin-like transcripts (NILTs) have been identified from salmon, trout and carp. NILTs typically encode activating or inhibitory transmembrane receptors with extracellular immunoglobulin (Ig) domains. Although predicted to provide some level of immune recognition in ray-finned fish, we currently lack a definitive framework of NILT diversity, thereby challenging our ability to understand the evolutionary origin and specific function of these genes. In order to better understand the diversity of NILT genes and their possible roles in immune function, we identified 5 NILT loci in the Atlantic salmon (Salmo salar) genome, defined 86 NILT Ig domains within a 3 Mbp region of zebrafish (Danio rerio) chromosome 1, and described 41 NILT Ig domains as part of an alternate haplotype for this same genomic region. We then identified transcripts that define 43 different NILT genes and reflect an unprecedented range of sequence diversity and combinatorial diversity of Ig do...
A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of th... more A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the ...
Proceedings of the National Academy of Sciences, 2016
Significance Antigen presentation genes are exceptionally polymorphic, enhancing immune defense. ... more Significance Antigen presentation genes are exceptionally polymorphic, enhancing immune defense. Polymorphism within additional components of the MHC pathway, particularly the antigen processing genes, may also shape immune responses. Using transcriptome, exome, and whole-genome sequencing to examine immune gene variation in zebrafish, we uncovered several antigen processing genes not found in the reference genome clustered within a deeply divergent haplotype of the core MHC locus. Our data provide evidence that these previously undescribed antigen processing genes retain ancient alternative sequence lineages, likely derived during the formation of the adaptive immune system, and represent the most divergent collection of antigen processing and presentation genes yet identified. These findings offer insights into the evolution of vertebrate adaptive immunity.
Cooley’s Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin ... more Cooley’s Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This study reports novel preclinical mouse models of CA that survive solely on human fetal hemoglobin at birth and are blood transfusion dependent for life upon completion of their human fetal to adult hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse αglobin genes with human α globin and the adult mouse β globin genes with a delayed switching human γβ° or γδβ° globin gene cassettes. The nonfunctional human β° globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous humanized CA mice express 100% human hemoglob...
Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin ge... more Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expres...
During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucl... more During erythropoiesis, hemoglobin (Hb) synthesis increases from early progenitors to mature enucleated erythrocytes. Although Hb is one of the most extensively studied proteins, the role of Hb in erythroid lineage commitment, differentiation, and maturation remains unclear. In this study, we generate mouse embryos and embryonic stem (ES) cells with all of the adult α and β globin genes deleted (Hb Null). While Hb Null embryos die in midgestation, adult globin genes are not required for primitive or definitive erythroid lineage commitment. In vitro differentiation of Hb Null ES cells generates viable definitive proerythroblasts that undergo apoptosis upon terminal differentiation. Surprisingly, all stages of Hb Null-derived definitive erythroblasts develop normally in vivo in chimeric mice, and Hb Null erythroid cells undergo enucleation to form reticulocytes. Free heme toxicity is not observed in Hb Null-derived erythroblasts. Transplantation of Hb Null-derived bone marrow cells pro...
A preclinical humanized mouse model of β thalassemia major or Cooley anemia (CA) was generated by... more A preclinical humanized mouse model of β thalassemia major or Cooley anemia (CA) was generated by targeted gene replacement of the mouse adult globin genes in embryonic stem cells. The mouse adult α and β globin genes were replaced with adult human α globin genes (α2α1) and a human fetal to adult hemoglobin (Hb)–switching cassette (γHPFHδβ0), respectively. Similar to human infants with CA, fully humanized mice survived postnatally by synthesizing predominantly human fetal Hb, HbF (α2γ2), with a small amount of human minor adult Hb, HbA2 (α2δ2). Completion of the human fetal to adult Hb switch after birth resulted in severe anemia marked by erythroid hyperplasia, ineffective erythropoiesis, hemolysis, and death. Similar to human patients, CA mice were rescued from lethal anemia by regular blood transfusion. Transfusion corrected the anemia and effectively suppressed the ineffective erythropoiesis, but led to iron overload. This preclinical humanized animal model of CA will be useful ...
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