![]() Two positive clones were selected for the blastocyst (C57BL/6) injection. The targeting vector was linearized and electroporated into 129/sv mouse ES cells, and subsequently screened by Southern blot ( Figure 1B). Because the endogenous ATG was disrupted, the Tet2:nlacZ/nGFP is also a heterozygous null for Tet2 ( Tet2 +/−) after the recombination. The nlacZ/nGFP is expressed under the control of the endogenous Tet2 promoter. The 5′ and 3′ arms of the targeting vector were amplified on 129/sv mouse genomic DNA. 16-21 The development of any type of tumor at an elevated frequency in such genetically altered mice adds persuasive evidence to support the candidacy of a gene as a tumor suppressor gene.Ī nuclear ß-galactosidase ( nlacZ) flanked by 2 loxP sites followed by a nuclear H2B-GFP ( nGFP) and neomycin ( Neo) was inserted 6 bp upstream of Tet2 start codon (cassette map: loxP-lacZ-polyA-loxP-H2B-GFP-polyA-FRT-Neo-FRT endogenous ATG was disrupted Figure 1A). 15 Almost all of the well studied tumor suppressor genes have been knocked out in the germ line of an inbred mouse strain, such as p53, NF1, AML1, APC, RB, and VHL. The ability to inactivate (“knock out”) candidate tumor suppressor genes in the mouse germ line provides a powerful tool for validating candidate tumor suppressor genes. 10-14 Therefore, TET2 might act as a tumor suppressor gene by regulating DNA methylation and epigenetic control of gene expression at critical loci important for myelopoiesis and leukemogenesis. 8, 9 All 3 paralogs share a highly homologous catalytic domain catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), which could epigenetically regulate gene expression by altering methylation-driven gene silencing. TET2 belongs to a 3-member family that also includes TET1 and TET3 TET1 was originally identified as a partner for the MLL gene within t(10 11)(p12 q23) translocations in AML. These data indicate that Tet2-deficient mice recapitulate patients with myeloid malignancies, implying that Tet2 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Furthermore, transplantation of Tet2 −/−, but not wild-type (WT) or Tet2 +/− BM cells, led to increased WBC counts, monocytosis, and splenomegaly in WT recipient mice. Approximately 1/3 of Tet2 −/− and 8% of Tet2 +/− mice died within 1 year of age because of the development of myeloid malignancies resembling characteristics of CMML, MPD-like myeloid leukemia, and MDS. A competitive reconstitution assay revealed that Tet2 −/− LSK cells had an increased hematopoietic repopulating capacity with an altered cell differentiation skewing toward monocytic/granulocytic lineages. The Tet2 −/− mice contained an increased Lin −Sca-1 +c-Kit + (LSK) cell pool before the development of myeloid malignancies. Deletion of Tet2 in mice led to dramatic reduction in the 5-hydroxymethylcytosine levels and concomitant increase in the 5-methylcytosine levels in the genomic DNA of BM cells. To study the function of TET2 in vivo, we generated a Tet2 knock out mouse model. However, little is known regarding the biological function of TET2 and its role in the pathogenesis of myeloid malignancies. TET2 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies including MDS, CMML, MPN, and AML.
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