| BackgroundIsocitrate Dehydrogenase (IDHs) are important in driving oxidative decarboxylation of isocitrate to a-ketoglutarate(a-KG) in the citric acid cycle. Isocitrate dehydrogenase catalyzes the first oxidative and decarboxylation steps in the citric acid cycle comprising a family of enzymes that convert isocitrate into a-ketoglutarate, rate-limiting step of Kerbs cycle. There are two families in IDH. The homodimeric enzymes IDH1 and IDH2 are NADP depedent, whereas the multitetrameric IDH3 composed of three subunits is NAD dependent. NAD-dependent IDH called idh3 is localized to the mitochondrial matrix. IDH3 forms a heteroteramer including two a subunits, one β subunit and one y subunit, and IDH3a play an important role in carcinogenesis. NADP-dependent IDH includes IDH1 which resides in cytosol and peroxisome and IDH2 which resides in mitochondrial matrix.idhl gene is located on the chromosome 9 of zebrafish. There are two transcripts in the idhl gene in ensembl database, they are idhl-201 and idhl-202. There are 9 exons in the idh1-201, the length of the transcript is 1995bp, and coding 429 amino acid.10 exons are on the idhl-202. The length of it is 1977bp, coding 423 amino acid. idh2 gene is located on the chromosome 18 of zebrafish. Two transcripts are on the idh2 gene in ensembl database. they are idh2-201 and idh2-202. There are 24 exons in the idh2-201, the length of the transcript is 1650bp, and coding 450 amino acid.16 exons are on the idh2-202. The length of it is 1641bp, coding 447 amino acid. idh3 gene is located on the chromosome 23 of zebrafish. Three transcripts are on the idh3 gene in ensembl database. they are idh3-201, idh3-003 and idh3-001.There are 13 exons in the idh3-201, the length of the transcript is 2475bp, and coding 391 amino acid.14 exons are on the idh3-003. The length of it is 2470bp, coding 391 amino acid.5 exons are on the idh3-001. The length of it is 1565bp, coding 66 amino acid.IDH3 forms a heteroteramer including two α subunits, one β subunit and one γ subunit, and IDH3α play an important role in carcinogenesis, but idh3 gene mutations have not been reported in terms of hematopoietic in human, So in this article idh3 gene is not the focus of our research. Since the milestone discovery of idh1 mutation in active sites of R132 and analogous amino acid (R172) of the idh2 gene in Glioblastoma Multiforme patients, continuous reports have described idh mutations as a root cause of human hematopoiesis disease, such as Myelodysplastic syndromes(MDS), Myeloproliferative neoplasms (MPN), Chronic myelomonocytic leukaemia (CMML), AML and lymphoid malignancies. idh1 and idh2 gene mutatation are heterozygous in general. IDH 1/2 mutations as the conformational change lead to decrease of α-KG and elevated level of D-2-HG. R132H IDH1 mutation dominantly inhibits the wild-type IDH1 by forming a catalytically inactive heterodimer, resulted in a decrease of cellular α-KG and elevated HIF-1αprotein levels. α-KG is required for EglN (also called PHD) family, enzymes that hydroxylate and promote the degradation of hypoxia-inducible factor1a (HIF-1α). Metabolic imbalances devotes to unusual high level of HIF-1α, which is required for survival maintenance of chronic myeloid leukemia stem cells and promotes process of tumor. Furthermore, IDH1 knockdown induced the expression of HIF-1α target genes including glucose transporter 1 (Glutl), vascular endothelial growth factor (VEGF), and phosphoglycerate kinase (PGK1). Elevated plasma 2-hydroxyglutarate in AML also is associate with severe renal impairment.D-2HG becomes ideally an optimal biomarker with IDH mutations.IDH mutations involved in epigenetic regulation can promote DNA and histone demethylation, which could contribute to hematopoiesis diseases.Ten-eleven-translocation (TET) molecules participate in the conversion of 5mC to 5hmC in zebrafish embryos. deletion of TET2 leads to erythrocyte dysplasia and anemia Idh mutations impair tet2 role in maintain the methylation level of hematopoietic related genes. IDH2 mutant leukemia mice displayed a massive reduction in 5hmC levels consistent with their ability to inhibit TET proteins. By comparing mouse,zebrafish and human IDH gene, we find their genes are very conservative. for example, the site of 132 locus in IDH1,140 and 172 sites in IDH2. Multiple research involved in study on cytology(cellular context), mouse models employed by knock-in idhl and idh2 mutation(idh mutations) in conservative sites and clinical cases with hematopoietic disease have well highlighted impaired hematopoiesis caused by idh mutations as oncogene. But there is not a good model to research blood disease by idh mutation.Taking into consideration the biological advantage of zebrafish, it is a good way to answer what lead to the abnormal hematopoiesis. Here we want to knock out the idhl and idh2 genes of zebrafish, screening the mutation, then we detect lymphocyte cells, marrow cells and erythrocyte cells.Gene knockout is indispensable to prove gene function, and is an important means of gene engineering and gene therapy. Common knockout method are Suicide plasmid, RNAi, ZFN, but there is a low efficiency by Suicide plasmid. Knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects, and provides only temporary inhibition of gene function. These restrictions impede researchers ability to link directly phenotype to genotype and limit the practical application of RNAi technology. The design of ZFN depends on the upstream and downstream sequence and has high off-target, high cytotoxicity. Its application is in vitro, and transfusion to the patient.It is easy to cause immune response. So it is difficult to use ZFN to knock out gene. TALEN, a new gene knockout way, avoids some disadvantages that has been mention as above. This core technology - commonly referred to as’genome editing’-is based on the use of engineered nucleases composed of sequence-specific DNA-binding domains fused to a nonspecific DNA cleavage module 3 and 4. These chimeric nucleases enable efficient and precise genetic modifications by inducing targeted DNA double-strand breaks (DSBs) that stimulate the cellular DNA repair mechanisms, including error-prone nonhomologous end joining (NHEJ) and homology-directed repair (HDR).TALEs are naturally occurring proteins from the plant pathogenic bacteria genus Xanthomonas, and contain DNA-binding domains composed of a series of 33-35-amino-acid repeat domains that each recognizes a single base pair. TALE specificity is determined by two hypervariable amino acids that are known as the repeat-variable di-residues (RVDs) 12 and 13. Like zinc fingers, modular TALE repeats are linked together to recognize contiguous DNA sequences.The NIRVD has been reported to bind to A. The NG RVD has been reported to bind to T. The HD RVD has been reported to bind to C. The NN RVD has been reported to bind to G. The minimal TALE region required for high-affinity DNA binding and identify truncation variants that enable efficient cleavage in vitro when linked to the catalytic domain of Fokl. Knocking out gene by Talen mainly includes basic domain of FokI. Knocking out gene by Talen mainly includes basic step:1.determining the target, selecting two target that the length are 14 to 18 bp of the target gene. The length of the exon in the target genes 17 to 18 bp; 2. clone construction where TAL identify; 3. cloning the TAL into certain eukaryotic expression vector; 4. micro-injecting the zebrafish with recombination plasmids to knock out gene; 5. screening the mutation. When the expression vector is micro-injected into the zebrafish, the next step we will detect the knockout efficiency, screen the mutation.we can use the enzyme that we make a choice in designing the target to digest. when there is no suitable digestion site, we consider use the enzyme CEL-I. we can construct all gene vector through unit assembly, so it is possible to knock out every gene.We knock out the idh1/2 gene by TALEN in the paper, obtaining the mutation.Then we have a discuss whether different mutation type can lead to diferent blood diseases. Experiment will be conducted in four part:the experimental materials, the experimental method, the results and analysis, discussion.Objective:To screen idh1/2 gene knockout zebrafish related to blood disease, and construct transcription activator-like effector nuelease (TALEN) vectors targeting idh gene,then identify some phenotypic correlation to hematopoietic lineage.Methods:We construct TALEN vectors targeting zabrafish idh gene using unit assembly method and the in vitro-transcribed TALEN mRNAs were micro-injected into one-cell stage zebrafish embryos. The efficiency of TALEN was identified by injected embryos, and mutations of zebrafish were screened and confirmed the different types through PCR and enzyme digestion. Finally, identify the mutations of related hematopoietic phenotypes in zebrafish with whole-mount in situ hybridizatio -n,Real-time fluorescent quantitative PCR, giemsa staining, Sudan black B stain.Results:We successfully constructed correct targeting vectors by enzyme digestion and sequencing, and the gene knockout efficiency was good as we expected. We screened the mutant zebrafish and confirmed different types of idh gene mutations.But there are no some mutation types related to hemopoiesis. I think hematopoietic abnormalities by idh gene mutation may be caused by a variety of gene interaction results. We need to find some new genes interact with idh gene further.Conclusions:A idhl/2 gene knockout zebrafish model is successfully established, it can provide a good animal model for further research of hemopoiesis. Because the hematopoiesis is a complicated process, it is a challenge to understand haematogenous mechanism. We can only simulate the mutation site in human with blood disease through idh mutation zebrafish, and find some mechanism leading to blood disease like leukemia. |
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