Establishment Of FLT3/ITD Knock-in Model In K562Cell Line By TALEN Gene Editing Technology

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.