Zhu Xiaofan

利用CRISPR/Cas9n double nick系统构建人 DNAH2 基因敲除的U2OS细胞株

To study the biological function of DNAH2 (Homo sapiens dynein, axonemal, heavy chain 2) gene, we constructed human stable U2OS cell line of DNAH2 gene knockout through CRISPR/Cas9n double nick system. The A, B sgRNAs (Single guide RNA) and complementary strands were designed and synthesized. The double-stranded structures were formed during annealing, and connected with BbsⅠ cohesive ends-containing pX462 linear vector to construct the recombinant eukaryotic expression plasmids, including pX462-DNAH2-A and pX462-DNAH2-B. After the co-transfection of the two plasmids into U2OS cells, the addition of puromycin and limiting dilution method were used to obtain positive monoclonal cell line. Western blotting assay was then performed to detect the expression of DNAH2 protein, and PCR-sequencing technology was finally utilized to analyze the mutation feature. The results showed that A, B sgRNAs duplex was successfully inserted into pX462 vector, and DNAH2 protein was not expressed and DNAH2 gene suffered from the frame-shift mutation in U2OS-DNAH2-KO monoclonal cell line. These demonstrated that DNAH2 knockout U2OS stable cell line was successfully constructed through CRISPR/Cas9n double nick system, which providing a useful tool for the study of DNAH2 gene.

Biological characterization of MLL-NRIP3 fusion gene induced leukemia

Leukemia, originating from hematopoietic stem or progenitor cells, is a malignant proliferative tumor which seriously endangers human health and survival. It is the most common childhood malignancy with an increasing proportion of malignancy in adults. Acute myeloid leukemia (AML) involving MLL (mixed lineage leukemia, MLL-r ) translocation is recognized as an aggressive form of hematopoietic malignancies that exhibit a blockade of differentiation, disruption of genetic and epigenetic regulation, and poor prognosis. Although MLL-NRIP3 fusion gene has been reported recently in establishing leukemia mouse model and its effect on leukemia progression in MLL-r mouse model has also been demonstrated, its leukemia biological characteristics were still not well characterized. Here we investigate the basic biological phenotypes of two MLL-r mouse models ( MLL-AF9 and MLL-NRIP3 ), including: the survival of recipient mice transplanted with leukemia cells; the migration and adhesion abilities of leukocyte cells; the number and the function of leukemia stem cells (LSCs), cell cycle and cell apoptosis, and investigate whether the surface markers of LSCs reported previously are equally applicable to MLL-NRIP3 leukemia cells. The results showed that the survival time of MLL-NRIP3 recipient mice was longer than that of MLL-AF9 . The migration ability of MLL-NRIP3 leukemia cells was less capable than MLL-AF9 , while adhesion was stronger than MLL-AF9 cells. Cell cycle and cell apoptosis of two MLL-r leukemia cells basically had no difference. C olony - forming unit assay and limiting dilution assay both revealed that MLL-AF9 leukemia had a higher number of functional LSCs than MLL-NRIP3 . In summary, MLL-NRIP3 induced leukemia is a low LSCs-enriched MLL-r .

The mechanism of B cell acute lymphoblastic leukemia development

B cell acute lymphoblastic leukemia (B-ALL) is a common malignant disease in children. Gene mutations in transcriptional factors and signal transduction molecules result in differentiation block and abnormal proliferation of B lymphoid progenitor cells. Children with B-ALL generally have a good prognosis, but about 20% of the pediatric patients and 60% adult patients still suffer from the high risk of relapse. Better understanding of the mechanism of leukemogenesis leads to significant improvement in treatment of ALL patients. Gene expression analysis and whole genome sequencing technologies have identified a number of novel gene mutations, but the roles of those mutations in leukemogenesisis remain unclear. This review mainly summarizes the so far identified cooperating gene mutations related to cell development and differentiation, epigenetic regulation, cell cycle and apoptosis, signal pathway, non-coding RNA, and leukemia clone evolution, some of which may be tightly associated with the relapse of B-ALL. In addition, we also review mouse models of B-ALL which may be useful for study of pathogenesis, risk classification, and design of future therapy.