利用基因编辑技术TALEN建立K562细胞系FLT3/ITD敲入模型
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摘要
类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease, TALEN)是人工构建的序列特异性核酸内切酶的一种,它能够识别并切割特定的DNA靶序列,造成双链断裂,并引起基因组结构的定点改变。它2010年底成功应用于基因打靶,很快成为一种比锌指核酸酶(Zinc-finger nuclease, ZFN)更容易设计、特异性更高和毒性更低的人工核酸内切酶。本文将用举例的方法从TALEN的设计、构建及六种活性或突变效率检测方法来建立并完善TALEN平台,并从方法的难易程度及优缺点进行评估六种突变率检测方法和合适的应用范围。通过UA (Unit Assembly,单元组装)和Golden Gate Vector based Assembly方法合成TALEN质粒,并比较酶切,SSA检测系统,T7E1酶切加毛细血管电泳检测,加药T在检测以及单克隆检测等方法,比较构建及筛选的优缺点。我们成功构建了TANLEN质粒,并建立了较多的检测TALEN切割效果的方法,比较了各种方法的优缺点,找到合适的评估体系。我们推荐T7E1酶切试验作为早期筛选,选择最佳的TALEN质粒；切割双荧光报告系统可以作为后期单克隆筛选的重要的富集系统。
FLT3/ITD突变是预测AML复发最重要的危险因素,能引起受体酪氨酸激酶显著而持久的活化,促进细胞增殖并抵抗凋亡。然而酪氨酸激酶抑制剂通常只能取得有限的血液学缓解,不能延长患者生存。因此,筛选FLT3/ITD突变促进白血病转化、加速疾病进展的关键基因或通路具有重要的理论和现实意义。然而FLT3/ITD突变常附加于其他具有表达谱特征的重现性遗传学异常,会对ITD突变相关基因或通路的筛选带来影响。在本次实验组,我们利用TALEN技术在白血病细胞株K562中的FLT3基因区引入或修正ITD突变,并分别从基因水平、表达水平、细胞行为学包括细胞增殖、凋亡、周期、等几个方面,以及下游基因的变化证实,该模型不仅在基因水平上有ITD的插入,同时也表达正确形式的FLT3/ITD剪切体,并具备相应的功能,引起了细胞行为学的相关变化。因此,我们成功建立了K562细胞系FLT3/ITD敲入模型。该模型有助于筛选出FLT3/ITD突变促进白血病转化、加速疾病进展的关键基因或信号通路,为解析FLT3-ITD突变的调控网络和分子机制,探寻潜在的治疗靶点提供参考。
正常的FLT3在维持造血系统的正常功能有重要的作用和意义。各种恶性血液病均有有不同程度的表达FLT3。大量的AML的样本观察到FLT3基因转录水平的增加,而这增加表达也可能有助于FLT3的磷酸化及其通路的激活。恶性血液病中,FLT3表达量高的患者具有更糟的OS和更低的无事件生存率。因此,了解FLT3在恶性血液病中的作用是非常重要。本文利用TALEN技术在K562上建立FLT3半敲除模型,通过比较半敲模型机野生型的K562细胞的细胞行为学变化以及效应相关基因的变化,结果显示,半敲除FLT3基因是细胞增殖减弱,克隆形成能力下降,同时随着FLT3基因的下降,其下游相关基因BASP1,IGFBP2,MSI2,STON2,PROX1也随之下降,说明些基因与FLT3有明显的相关性。由此说明抑制FLT3可以抑制肿瘤细胞的增殖生长及克隆形成,同时抑制FLT3,也抑制BASP1,IGFBP2,MSI2,STON2,PROX1基因的表达。该模型可以帮助深入探讨FLT3在恶性血液病中的机制,同时也可为为寻找抑制FLT3基因新的靶点提供较好的平台。
Artificial designer nucleases targeting specific DNA sequences open up a new field for reverse genetics study. The construction of transcription activator-like effector nucleases (TALENs) is simpler with higher specificity and less toxicity than zinc-finger nucleases (ZFNs). This article detailed described design of TALEN, two kinds of TALEN plasmid construction method and six kinds of mutation detection method to establish and improve the TALEN platform. The two method including Unit Assembly unit assembly and Golden Gate Vector based Assembly wo μ Id be discribed. The five mutation screening systems included the digested resistance testing, SSA detection system, T7E1enzyme and capillary electrophoresis detection system, T plasmid detection system and single clone detection system. We successfμlly constructed TANLEN plasmid, and the establishment of the method of detection TALEN cutting resμlts more. After assessing the degree of difficμlty of the methods and compareing the advantages and disadvantages of mutation detection systems, we woμld like to give our advice of appropriate scope of application of all the six mutation screening system. We recommend T7E1enzyme and capillary electrophoresis detection system as an early detection, and choose the best TALEN plasmid; dual fluorescent reporter system can be used as a post-screening monoclonal enrichment system.
FLT3/ITD mutation serves as a risk factor predicting relapse for AML patients. The sufficient and sustained activation of receptor tyrosine kinase induced by FLT3/ITD promotes the proliferation of leukemia cells and resists apoptosis. The screening of FLT3/ITD-related genes or pathways which promote leukemogenesis and accelerate leukemia progression has not only theoretical significance, but practical value. However, the characteristics of gene expression profiling from other concomitant genetic lesions will impede the screening of regμlatory genes or pathways related to FLT3-ITD. Therefore, to reveal the differentially expressed genes derived from ITD mutation, we plan to establish engineered leukemia cell models by TALEN in FLT3gene locus. In this experiment, we used TALEN technology to knock in ITD mutation into the FLT3gene region in the leukemia cell line K562. Then, we did tests in gene level, expression levels and cell behavior, including cell proliferation, apoptosis, cycle, as well as the changes in downstream genes. we confirmed that the FLT3/ITD knock-in K562cell model had the expected gene change in DNA level, and expressed the correct form of FLT3/ITD in mRNA level. Comparing wild-type K562cell line, the downstream genes in K562ITD/wt model was expected changed and FLT3level remained unchange. On the point of cell behavior, K562ITD/wt model also possessed higher cell survive rate, more S phase of cell cycle, and less apoptosis than wild-type K562cell line when at a low concentration of FBS, which was the expected function change by FLT3/ITD. Therefore, we successfμlly established FLT3/ITD knock-in K562cell line model.This model coμld help us to understand much more about the FLT3/ITD in AML,and resolve the regμlatory network and provide novel therapeutic targets. Normal FLT3gene has an important role in maintaining the normal function of the hematopoietic system and significance. A variety of hematologic malignancies have different levels of expression of FLT3. The AML sample observed increase in the level of the FLT3gene transcription, which increased expression of FLT3phosphorylation pathway may also contribute. Hematologic malignancies in patients with high FLT3expression levels with worse OS and lower event-free survival. Therefore, understanding the role of FLT3in hematologic malignancies is very important. We used the TALEN technology create FLT3gene hyploinsufficiency knockout model in K562cell line。 By comparing the model of wild type K562cells, cell behavior of half knockout FLT3K562cell line shows cell proliferation and colony forming capacity decreased. At the same time with the decline of the FLT3gene, its downstream gene BASP1, in IGFBP2, MSI2STON2PROX1also fall down, which means a significant correlation between these genes and FLT3gene. It showed inhibition of FLT3can inhibit the growth of tumor cell proliferation and colony formation, while inhibition of FLT3also inhibited BASP1, IGFBP2, MSI2, STON2, PROX1gene expression. The model can help to further explore the mechanism of FLT3in hematologic malignancies, but also provide a better platform for looking for a new target for inhibition of FLT3gene.
FLT3/ITD突变是预测AML复发最重要的危险因素,能引起受体酪氨酸激酶显著而持久的活化,促进细胞增殖并抵抗凋亡。然而酪氨酸激酶抑制剂通常只能取得有限的血液学缓解,不能延长患者生存。因此,筛选FLT3/ITD突变促进白血病转化、加速疾病进展的关键基因或通路具有重要的理论和现实意义。然而FLT3/ITD突变常附加于其他具有表达谱特征的重现性遗传学异常,会对ITD突变相关基因或通路的筛选带来影响。在本次实验组,我们利用TALEN技术在白血病细胞株K562中的FLT3基因区引入或修正ITD突变,并分别从基因水平、表达水平、细胞行为学包括细胞增殖、凋亡、周期、等几个方面,以及下游基因的变化证实,该模型不仅在基因水平上有ITD的插入,同时也表达正确形式的FLT3/ITD剪切体,并具备相应的功能,引起了细胞行为学的相关变化。因此,我们成功建立了K562细胞系FLT3/ITD敲入模型。该模型有助于筛选出FLT3/ITD突变促进白血病转化、加速疾病进展的关键基因或信号通路,为解析FLT3-ITD突变的调控网络和分子机制,探寻潜在的治疗靶点提供参考。
正常的FLT3在维持造血系统的正常功能有重要的作用和意义。各种恶性血液病均有有不同程度的表达FLT3。大量的AML的样本观察到FLT3基因转录水平的增加,而这增加表达也可能有助于FLT3的磷酸化及其通路的激活。恶性血液病中,FLT3表达量高的患者具有更糟的OS和更低的无事件生存率。因此,了解FLT3在恶性血液病中的作用是非常重要。本文利用TALEN技术在K562上建立FLT3半敲除模型,通过比较半敲模型机野生型的K562细胞的细胞行为学变化以及效应相关基因的变化,结果显示,半敲除FLT3基因是细胞增殖减弱,克隆形成能力下降,同时随着FLT3基因的下降,其下游相关基因BASP1,IGFBP2,MSI2,STON2,PROX1也随之下降,说明些基因与FLT3有明显的相关性。由此说明抑制FLT3可以抑制肿瘤细胞的增殖生长及克隆形成,同时抑制FLT3,也抑制BASP1,IGFBP2,MSI2,STON2,PROX1基因的表达。该模型可以帮助深入探讨FLT3在恶性血液病中的机制,同时也可为为寻找抑制FLT3基因新的靶点提供较好的平台。
Artificial designer nucleases targeting specific DNA sequences open up a new field for reverse genetics study. The construction of transcription activator-like effector nucleases (TALENs) is simpler with higher specificity and less toxicity than zinc-finger nucleases (ZFNs). This article detailed described design of TALEN, two kinds of TALEN plasmid construction method and six kinds of mutation detection method to establish and improve the TALEN platform. The two method including Unit Assembly unit assembly and Golden Gate Vector based Assembly wo μ Id be discribed. The five mutation screening systems included the digested resistance testing, SSA detection system, T7E1enzyme and capillary electrophoresis detection system, T plasmid detection system and single clone detection system. We successfμlly constructed TANLEN plasmid, and the establishment of the method of detection TALEN cutting resμlts more. After assessing the degree of difficμlty of the methods and compareing the advantages and disadvantages of mutation detection systems, we woμld like to give our advice of appropriate scope of application of all the six mutation screening system. We recommend T7E1enzyme and capillary electrophoresis detection system as an early detection, and choose the best TALEN plasmid; dual fluorescent reporter system can be used as a post-screening monoclonal enrichment system.
FLT3/ITD mutation serves as a risk factor predicting relapse for AML patients. The sufficient and sustained activation of receptor tyrosine kinase induced by FLT3/ITD promotes the proliferation of leukemia cells and resists apoptosis. The screening of FLT3/ITD-related genes or pathways which promote leukemogenesis and accelerate leukemia progression has not only theoretical significance, but practical value. However, the characteristics of gene expression profiling from other concomitant genetic lesions will impede the screening of regμlatory genes or pathways related to FLT3-ITD. Therefore, to reveal the differentially expressed genes derived from ITD mutation, we plan to establish engineered leukemia cell models by TALEN in FLT3gene locus. In this experiment, we used TALEN technology to knock in ITD mutation into the FLT3gene region in the leukemia cell line K562. Then, we did tests in gene level, expression levels and cell behavior, including cell proliferation, apoptosis, cycle, as well as the changes in downstream genes. we confirmed that the FLT3/ITD knock-in K562cell model had the expected gene change in DNA level, and expressed the correct form of FLT3/ITD in mRNA level. Comparing wild-type K562cell line, the downstream genes in K562ITD/wt model was expected changed and FLT3level remained unchange. On the point of cell behavior, K562ITD/wt model also possessed higher cell survive rate, more S phase of cell cycle, and less apoptosis than wild-type K562cell line when at a low concentration of FBS, which was the expected function change by FLT3/ITD. Therefore, we successfμlly established FLT3/ITD knock-in K562cell line model.This model coμld help us to understand much more about the FLT3/ITD in AML,and resolve the regμlatory network and provide novel therapeutic targets. Normal FLT3gene has an important role in maintaining the normal function of the hematopoietic system and significance. A variety of hematologic malignancies have different levels of expression of FLT3. The AML sample observed increase in the level of the FLT3gene transcription, which increased expression of FLT3phosphorylation pathway may also contribute. Hematologic malignancies in patients with high FLT3expression levels with worse OS and lower event-free survival. Therefore, understanding the role of FLT3in hematologic malignancies is very important. We used the TALEN technology create FLT3gene hyploinsufficiency knockout model in K562cell line。 By comparing the model of wild type K562cells, cell behavior of half knockout FLT3K562cell line shows cell proliferation and colony forming capacity decreased. At the same time with the decline of the FLT3gene, its downstream gene BASP1, in IGFBP2, MSI2STON2PROX1also fall down, which means a significant correlation between these genes and FLT3gene. It showed inhibition of FLT3can inhibit the growth of tumor cell proliferation and colony formation, while inhibition of FLT3also inhibited BASP1, IGFBP2, MSI2, STON2, PROX1gene expression. The model can help to further explore the mechanism of FLT3in hematologic malignancies, but also provide a better platform for looking for a new target for inhibition of FLT3gene.
引文
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2. Bibikova M, Golic M, Golic KG, et al. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics,2002; 161(3): 1169-75.
3. Moscou MJ and Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science,2009; 326(5959):1501.
4. Boch J, Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type Ⅲ effectors. Science,2009; 326(5959):1509-12.
5. Method of the Year 2011. Nat Methods,2012; 9(1):1.
6. Boch J and Bonas U. Xanthomonas AvrBs3 family-type Ⅲ effectors:discovery and function. Annu Rev Phytopathol,2010; 48:419-36.
7. Li L, Piatek MJ, Atef A, et al. Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification. Plant Mol Biol,2012; 78(4-5):407-16.
8. Zhang F, Cong L, Lodato S, et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol,2011; 29(2): 149-53.
9. Sanjana NE, Cong L, Zhou Y, et al. A transcription activator-like effector toolbox for genome engineering. Nat Protoc,2012; 7(1):171-92.
10. Li T, Huang S, Zhao X, et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res, 2011; 39(14):6315-25.
11. Briggs AW, Rios X, Chari R, et al. Iterative capped assembly:rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res,2012; 40(15):e117.
12. Li T, Huang S, Jiang WZ, et al. TAL nucleases (TALNs):hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res,2011; 39(1): 359-72.
13. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol,2011; 29(8):699-700.
14. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol,2011; 29(8):697-8.
15. Reyon D, Tsai SQ, Khayter C, et al. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol,2012; 30(5):460-5.
16. Schmid-Burgk JL, Schmidt T, Kaiser V, et al. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat Biotechnol,2013; 31(1):76-81.
1. Schlenk RF, Dohner K, Krauter J, et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med,2008; 358(18): 1909-18.
2. Stirewalt DL and Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer,2003; 3(9):650-65.
3. B u llinger L, Dohner K, Kranz R, et al. An FLT3 gene-expression signature predicts clinical outcome in normal karyotype AML. Blood,2008; 111(9):4490-5.
4. Garzon R, Volinia S, Liu CG, et al. MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood,2008; 111(6):3183-9.
5. Whitman SP, Maharry K, Radmacher MD, et al. FLT3 internal tandem duplication associates with adverse outcome and gene-and microRNA-expression signatures in patients 60 years of age or older with primary cytogenetically normal acute myeloid leukemia:a Cancer and Leukemia Group B study. Blood,2010; 116(18):3622-6.
6. Dohner H and Gaidzik VI. Impact of genetic features on treatment decisions in AML. Hematology Am Soc Hematol Educ Program,2011; 2011:36-42.
7. Kindler T, Lipka DB, and Fischer T. FLT3 as a therapeutic target in AML:still challenging after all these years. Blood,2010; 116(24):5089-102.
8. Christensen JL and Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation:a simple method to isolate long-term stem cells. Proc Natl Acad Sci USA,2001; 98(25):14541-6.
9. Kikushige Y, Yoshimoto G, Miyamoto T, et al. Human Flt3 is expressed at the hematopoietic stem cell and the gran μ locyte/macrophage progenitor stages to maintain cell survival. J Immunol,2008; 180(11):7358-67.
10. Boyer SW, Schroeder AV, Smith-Berdan S, et al. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells. Cell Stem Cell,2011; 9(1):64-73.
11. Sitnicka E, Buza-Vidas N, Larsson S, et al. Human CD34+ hematopoietic stem cells capable of m μ ltilineage engrafting NOD/SCID mice express flt3:distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells. Blood,2003; 102(3):881-6.
12. Mackarehtschian K, Hardin JD, Moore KA, et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity,1995; 3(1):147-61.
13. Gale RE, Green C, Allen C, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young ad μ lt patients with acute myeloid leukemia. Blood,2008; 111(5): 2776-84.
14. Zhang S, Fukuda S, Lee Y, et al. Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling. J Exp Med,2000; 192(5):719-28.
15. Valk PJ, Verhaak RG, Beijen MA, et al. Prognostically usef μ 1 gene-expression profiles in acute myeloid leukemia. N Engl J Med,2004; 350(16):1617-28.
16. Lee BH, Tothova Z, Levine RL, et al. FLT3 mutations confer enhanced proliferation and survival properties to m μ ltipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell,2007; 12(4):367-80.
17. Kelly LM, Liu Q, Kutok JL, et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model. Blood,2002; 99(1):310-8.
18. Takahashi S. Downstream molecμlar pathways of FLT3 in the pathogenesis of acute myeloid leukemia:biology and therapeutic implications. J Hematol Oncol,2011; 4: 13.
19. Pemmaraju N, Kantarjian H, Ravandi F, et al. FLT3 inhibitors in the treatment of acute myeloid leukemia:the start of an era? Cancer,2011; 117(15):3293-304.
20. Levis M, Ravandi F, Wang ES, et al. Res μ Its from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Blood,2011; 117(12):3294-301.
21. Fischer T. Rethinking bioactivity of FLT3 inhibitors. Blood,2011; 117(12):3247-8.
22. von Bubnoff N, Engh RA, Aberg E, et al. FMS-like tyrosine kinase 3-internal tandem duplication tyrosine kinase inhibitors display a nonoverlapping profile of resistance mutations in vitro. Cancer Res,2009; 69(7):3032-41.
23. Mali RS, Ramdas B, Ma P, et al. Rho kinase reg μ lates the survival and transformation of cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL. Cancer Cell,2011; 20(3):357-69.
24. Chen LS, Redkar S, Taverna P, et al. Mechanisms of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia. Blood,2011; 118(3):693-702.
25. Adam M, Pogacic V, Bendit M, et al. Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL. Cancer Res, 2006; 66(7):3828-35.
26. Levis M, Allebach J, Tse KF, et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood,2002; 99(11):3885-91.
27. Mizuki M, Fenski R, Halfter H, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood,2000; 96(12):3907-14.
28. Grundler R, Miething C, Thiede C, et al. FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model. Blood,2005; 105(12):4792-9.
29. Lumk μ l R, Gorin NC, Malehorn MT, et al. Human AML cells in NOD/SCID mice: engraftment potential and gene expression. Leukemia,2002; 16(9):1818-26.
30. Figueroa ME, Lugthart S, Li Y, et al. DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemia. Cancer Cell,2010; 17(1): 13-27.
31. Li L, Piloto O, Nguyen HB, et al. Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model. Blood,2008; 111(7):3849-58.
32. Bogdanove AJ and Voytas DF. TAL effectors:customizable proteins for DNA targeting. Science,2011; 333(6051):1843-6.
33. Miller JC, Tan S, Qiao G, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol,2011; 29(2):143-8.
34. Hockemeyer D, Wang H, Kiani S, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol,2011; 29(8):731-4.
35. Bedell VM, Wang Y, Campbell JM, et al. In vivo genome editing using a high-efficiency TALEN system. Nature,2012; 491(7422):114-8.
36. Zu Y, Tong X, Wang Z, et al. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods,2013; 10(4):329-31.
1. Markovic A, MacKenzie KL, and Lock RB. FLT-3:a new focus in the understanding of acute leukemia. Int J Biochem Cell Biol,2005; 37(6):1168-72.
2. Rosnet O, Schiff C, Pebusque MJ, et al. Human FLT3/FLK2 gene:cDNA cloning and expression in hematopoietic cells. Blood,1993; 82(4):1110-9.
3. Rosnet O, Buhring HJ, Marchetto S, et al. Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia,1996; 10(2):238-48.
4. Gabbianelli M, Pelosi E, Montesoro E, et al. Mμlti-level effects of flt3 ligand on human hematopoiesis:expansion of putative stem cells and proliferation of gran μ lomonocytic progenitors/monocytic precursors. Blood,1995; 86(5):1661-70.
5. Lyman SD and Jacobsen SE. c-kit ligand and Flt3 ligand:stem/progenitor cell factors with overlapping yet distinct activities. Blood,1998; 91 (4):1101-34.
6. Rusten LS, Lyman SD, Veiby OP, et al. The FLT3 ligand is a direct and potent stim u lator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro. Blood,1996; 87(4):1317-25.
7. Shah AJ, Smogorzewska EM, Hannum C, et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38- cells and maintains progenitor cells in vitro. Blood,1996; 87(9):3563-70.
8. Ray RJ, Paige CJ, Furlonger C, et al. Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7. Eur J Immunol,1996; 26(7):1504-10.
9. Veiby OP, Lyman SD, and Jacobsen SE. Combined signaling through interleukin-7 receptors and flt3 but not c-kit potently and selectively promotes B-cell commitment and differentiation from uncommitted murine bone marrow progenitor cells. Blood,1996; 88(4):1256-65.
10. Namikawa R, Muench MO, de Vries JE, et al. The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro. Blood,1996; 87(5):1881-90.
11. Armstrong SA, Kung AL, Mabon ME, et al. Inhibition of FLT3 in MLL. Validation of a therapeutic target identified by gene expression based classification. Cancer Cell,2003; 3(2):173-83.
12. Ozeki K, Kiyoi H, Hirose Y, et al. Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia. Blood,2004; 103(5):1901-8.
13. Zheng R, Levis M, Piloto O, et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells. Blood,2004; 103(1):267-74.
14. Choudhary C, Schwable J, Brandts C, et al. AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations. Blood,2005; 106(1):265-73.
15. Kuchenbauer F, Kern W, Schoch C, et al. Detailed analysis of FLT3 expression levels in acute myeloid leukemia. Haematologica,2005; 90(12):1617-25.
16. Armstrong SA, Staunton JE, Silverman LB, et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet, 2002; 30(1):41-7.
17. Levis M and Small D. FLT3 tyrosine kinase inhibitors. Int J Hematol,2005; 82(2): 100-7.
18. Scott E, Hexner E, Perl A, et al. Targeted signal transduction therapies in myeloid malignancies. Curr Oncol Rep,2010; 12(6):358-65.
19. Stone RM, DeAngelo DJ, Klimek V, et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molec μ le FLT3 tyrosine kinase inhibitor, PKC412. Blood,2005; 105(1):54-60.
20. Huang L, Zhou K, Yang Y, et al. FLT3-ITD-associated gene-expression signatures in NPM1-mutated cytogenetically normal acute myeloid leukemia. Int J Hematol, 2012; 96(2):234-40.
21. Method of the Year 2011. Nat Methods,2012; 9(1):1.
22. Levis M, Allebach J, Tse KF, et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood,2002; 99(11):3885-91.
23. Mizuki M, Fenski R, Halfter H, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood,2000; 96(12):3907-14.
24. Walters DK, Stoffregen EP, Heinrich MC, et al. RNAi-induced down-reg μ lation of FLT3 expression in AML cell lines increases sensitivity to MLN518. Blood, 2005; 105(7):2952-4.
1. Bibikova M, Golic M, Golic KG, et al. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics,2002; 161(3): 1169-75.
2. Moscou MJ and Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science,2009; 326(5959):1501.
3. Boch J, Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type Ⅲ effectors. Science,2009; 326(5959):1509-12.
4. Method of the Year 2011. Nat Methods,2012; 9(1):1.
5. Boch J and Bonas U. Xanthomonas AvrBs3 family-type Ⅲ effectors:discovery and function. Annu Rev Phytopathol,2010; 48:419-36.
6. Cong L, Zhou R, Kuo YC, et al. Comprehensive interrogation of natural TALE DNA-binding modμles and transcriptional repressor domains. Nat Commun,2012; 3:968.
7. Streubel J, Blucher C, Landgraf A, et al. TAL effector RVD specificities and efficiencies. Nat Biotechnol,2012; 30(7):593-5.
8. Zhang F, Cong L, Lodato S, et al. Efficient construction of sequence-specific TAL effectors for modμlating mammalian transcription. Nat Biotechnol,2011; 29(2): 149-53.
9. Sanjana NE, Cong L, Zhou Y, et al. A transcription activator-like effector toolbox for genome engineering. Nat Protoc,2012; 7(1):171-92.
10. Li T, Huang S, Zhao X, et al. Modμlarly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res, 2011; 39(14):6315-25.
11. Sander JD, Cade L, Khayter C, et al. Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol,2011; 29(8):697-8.
12. Li L, Piatek MJ, Atef A, et al. Rapid and highly efficient construction of TALE-based transcriptional regμlators and nucleases for genome modification. Plant Mol Biol,2012; 78(4-5):407-16.
13. Briggs AW, Rios X, Chari R, et al. Iterative capped assembly:rapid and scalable synthesis of repeat-modμle DNA such as TAL effectors from individual monomers. Nucleic Acids Res,2012; 40(15):e117.
14. Li T, Huang S, Jiang WZ, et al. TAL nucleases (TALNs):hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res,2011; 39(1): 359-72.
15. Huang P, Xiao A, Zhou M, et al. Heritable gene targeting in zebrafish using customized TALENs. Nat Biotechnol,2011; 29(8):699-700.
16. Miller JC, Tan S, Qiao G, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol,2011; 29(2):143-8.
17. Reyon D, Tsai SQ, Khayter C, et al. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol,2012; 30(5):460-5.
18. Schmid-Burgk JL, Schmidt T, Kaiser V, et al. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat Biotechnol,2013; 31(1):76-81.
19. Christensen JL and Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation:a simple method to isolate long-term stem cells. Proc Natl Acad Sci USA,2001; 98(25):14541-6.
20. Kikushige Y, Yoshimoto G, Miyamoto T, et al. Human Flt3 is expressed at the hematopoietic stem cell and the granμlocyte/macrophage progenitor stages to maintain cell survival. J Immunol,2008; 180(11):7358-67.
21. Boyer SW, Schroeder AV, Smith-Berdan S, et al. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells. Cell Stem Cell,2011; 9(1):64-73.
22. Sitnicka E, Buza-Vidas N, Larsson S, et al. Human CD34+ hematopoietic stem cells capable of mμltilineage engrafting NOD/SCID mice express flt3:distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells. Blood,2003; 102(3):881-6.
23. Mackarehtschian K, Hardin JD, Moore KA, et al. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity,1995; 3(1):147-61.
24. Dohner H and Gaidzik VI. Impact of genetic features on treatment decisions in AML. Hematology Am Soc Hematol Educ Program,2011; 2011:36-42.
25. Zhang S, Fukuda S, Lee Y, et al. Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling. J Exp Med,2000; 192(5):719-28.
26. Valk PJ, Verhaak RG, Beijen MA, et al. Prognostically usefμl gene-expression profiles in acute myeloid leukemia. N Engl J Med,2004; 350(16):1617-28.
27. Lee BH, Tothova Z, Levine RL, et al. FLT3 mutations confer enhanced proliferation and survival properties to mμltipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell,2007; 12(4):367-80.
28. Kelly LM, Liu Q, Kutok JL, et al. FLT3 internal tandem duplication mutations associated with human acute myeloid leukemias induce myeloproliferative disease in a murine bone marrow transplant model.Blood,2002; 99(1):310-8.
29. Takahashi S. Downstream molecμlar pathways of FLT3 in the pathogenesis of acute myeloid leukemia:biology and therapeutic implications. J Hematol Oncol,2011; 4: 13.
30. Kindler T, Lipka DB, and Fischer T. FLT3 as a therapeutic target in AML:still challenging after all these years. Blood,2010; 116(24):5089-102.
31. Pemmaraju N, Kantarjian H, Ravandi F, et al. FLT3 inhibitors in the treatment of acute myeloid leukemia:the start of an era? Cancer,2011; 117(15):3293-304.
32. Levis M, Ravandi F, Wang ES, et al. Resμlts from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Blood,2011; 117(12):3294-301.
33. Fischer T. Rethinking bioactivity of FLT3 inhibitors. Blood,2011; 117(12):3247-8.
34. von Bubnoff N, Engh RA, Aberg E, et al. FMS-like tyrosine kinase 3-internal tandem duplication tyrosine kinase inhibitors display a nonoverlapping profile of resistance mutations in vitro. Cancer Res,2009; 69(7):3032-41.
35. Mali RS, Ramdas B, Ma P, et al. Rho kinase regμlates the survival and transformation of cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL. Cancer Cell,2011; 20(3):357-69.
36. Chen LS, Redkar S, Taverna P, et al. Mechanisms of cytotoxicity to Pim kinase inhibitor, SGI-1776, in acute myeloid leukemia. Blood,2011; 118(3):693-702.
37. Adam M, Pogacic V, Bendit M, et al. Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL. Cancer Res, 2006; 66(7):3828-35.
38. Levis M, Allebach J, Tse KF, et al. A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo. Blood,2002; 99(11):3885-91.
39. Mizuki M, Fenski R, Halfter H, et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood,2000; 96(12):3907-14.
40. Grundler R, Miething C, Thiede C, et al. FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model. Blood,2005; 105(12):4792-9.
41. Lumkμl R, Gorin NC, Malehorn MT, et al. Human AML cells in NOD/SCID mice: engraftment potential and gene expression. Leukemia,2002; 16(9):1818-26.
42. Figueroa ME, Lugthart S, Li Y, et al. DNA methylation signatures identify biologically distinct subtypes in acute myeloid leukemia. Cancer Cell,2010; 17(1): 13-27.
43. Li L, Piloto O, Nguyen HB, et al. Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model. Blood,2008; 111(7):3849-58.
44. Bogdanove AJ and Voytas DF. TAL effectors:customizable proteins for DNA targeting. Science,2011; 333(6051):1843-6.
45. Gale RE, Green C, Allen C, et al. The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adμlt patients with acute myeloid leukemia. Blood,2008; 111(5):2776-84.
46. Greenblatt S, Li L, Slape C, et al. Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model. Blood,2012; 119(12):2883-94.
47. Rosnet O, Buhring HJ, Marchetto S, et al. Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia,1996; 10(2):238-48.
48. Hockemeyer D, Wang H, Kiani S, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol,2011; 29(8):731-4.
49. Bedell VM, Wang Y, Campbell JM, et al. In vivo genome editing using a high-efficiency TALEN system. Nature,2012; 491(7422):114-8.
50. Zu Y, Tong X, Wang Z, et al. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods,2013; 10(4):329-31.
51. Huang L, Zhou K, Yang Y, et al. FLT3-ITD-associated gene-expression signatures in NPM1-mutated cytogenetically normal acute myeloid leukemia. Int J Hematol, 2012; 96(2):234-40.
52. Walters DK, Stoffregen EP, Heinrich MC, et al. RNAi-induced down-regμlation of FLT3 expression in AML cell lines increases sensitivity to MLN518. Blood,2005; 105(7):2952-4.
53. Yokota S, Kiyoi H, Nakao M, et al. Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia,1997; 11(10):1605-9.
54. Gilliland DG and Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood,2002; 100(5):1532-42.
55. Kelly LM, Kutok JL, Williams IR, et al. PML/RARalpha and FLT3-ITD induce an APL-like disease in a mouse model. Proc Natl Acad Sci U S A,2002; 99(12): 8283-8.
56. Markovic A, MacKenzie KL, and Lock RB. FLT-3:a new focus in the understanding of acute leukemia. Int J Biochem Cell Biol,2005; 37(6):1168-72.
57. Rosnet O, Schiff C, Pebusque MJ, et al. Human FLT3/FLK2 gene:cDNA cloning and expression in hematopoietic cells. Blood,1993; 82(4):1110-9.
58. Gabbianelli M, Pelosi E, Montesoro E, et al. Mμlti-level effects of flt3 ligand on human hematopoiesis:expansion of putative stem cells and proliferation of granμlomonocytic progenitors/monocytic precursors. Blood,1995; 86(5):1661-70.
59. Lyman SD and Jacobsen SE. c-kit ligand and Flt3 ligand:stem/progenitor cell factors with overlapping yet distinct activities. Blood,1998; 91(4):1101-34.
60. Dosil M, Wang S, and Lemischka IR. Mitogenic signalling and substrate specificity of the Flk2/Flt3 receptor tyrosine kinase in fibroblasts and interleukin 3-dependent hematopoietic cells. Mol Cell Biol,1993; 13(10):6572-85.
61. Zhang S and Broxmeyer HE. Flt3 ligand induces tyrosine phosphorylation of gab1 and gab2 and their association with shp-2, grb2, and PI3 kinase. Biochem Biophys Res Commun,2000; 277(1):195-9.
62. Marchetto S, Fournier E, Beslu N, et al. SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor. Leukemia,1999; 13(9): 1374-82.
63. Lavagna-Sevenier C, Marchetto S, Birnbaum D, et al. FLT3 signaling in hematopoietic cells involves CBL, SHC and an unknown P115 as prominent tyrosine-phosphorylated substrates. Leukemia,1998; 12(3):301-10.
64. Stirewalt DL and Radich JP. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer,2003; 3(9):650-65.
65. Brasel K, Escobar S, Anderberg R, et al. Expression of the flt3 receptor and its ligand on hematopoietic cells. Leukemia,1995; 9(7):1212-8.
66. Takahashi S. Inhibition of the MEK/MAPK signal transduction pathway strongly impairs the growth of Flt3-ITD cells. Am J Hematol,2006; 81(2):154-5.
67. Hayakawa F, Towatari M, Kiyoi H, et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene,2000; 19(5):624-31.
68. Ray RJ, Paige CJ, Furlonger C, et al. Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7. Eur J Immunol,1996; 26(7):1504-10.
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