Progress of gene editing technologies and prospect in traditional Chinese medicine / 中国中药杂志

Gene editing is a kind of technologies that makes precise modification to the genome. It can be used to knock out/in and replace the specific DNA fragment, and make accurate gene editing on the genome level. The essence of the technique is the DNA sequence change with use of non homologous end link repair and homologous recombination repair, combined with specific DNA target recognition and endonuclease.This technology has wide range of development prospects and high application value in terms of scientific research, agriculture, medical treatment and other fields. In the field of gene therapy, gene editing technology has achieved cross-time success in cancers such as leukemia, genetic disorders such as hemophilia, thalassemia, multiple muscle nutritional disorders and retrovirus associated infectious diseases such as AIDS and other diseases. The preparation work for new experimental methods and animal models combined with gene editing technology is under rapid development and improvement. Laboratories around the world have also applied gene editing technique in prevention of malaria, organ transplantation, biological pharmaceuticals, agricultural breeding improvement, resurrection of extinct species, and other research areas. This paper summarizes the application and development status of gene editing technique in the above fields, and also preliminarily explores the potential application prospect of the technology in the field of traditional Chinese medicine, and discusses the present controversy and thoughts.

Genome-editing: focus on the off-target effects / 生物工程学报

Breakthroughs of genome-editing in recent years have paved the way to develop new therapeutic strategies. These genome-editing tools mainly include Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. However, off-target effects are still the major issue in genome editing, and limit the application in gene therapy. Here, we summarized the cause and compared different detection methods of off-targets.

Establishment of L-periaxin gene knock-out RSC96 cell line / 生物工程学报

Periaxin, a protein of noncompact myelin, is specifically expressed in the peripheral nervous system (PNS). There are two protein isoform L-periaxin and S-Periaxin by alternative splicing of periaxin gene, playing an important role in the initiation of myelin formation. So far, 18 different mutation sites in L-periaxin gene have been found to induce the peripheral demyelinating neurological charcot-marie-tooth diseases subtype 4F (CMT4F). The technique of activation of transcription activator-like effector nucleases (TALENS) was used to knock out the L-periaxin gene in RSC 96 cell line of Rattus. According to the design principle, the knock-out site of L-periaxin was assured to NLS domain of L-periaxin, which is target sequence of left and right arms of TALEN. The knock-out vectors of TALEN-L and TALEN-R were established and transfected into RSC96 cell. After puromycin screening, L-periaxin was knocked out successfully in RSC96 cell, which is confirmed by DNA sequence. The mutation efficiency is 21.6%. S-periaxin, not L-periaxin can be detected by Western blotting in L-periaxin gene knock-out RSC96 cell. The cell growth rate was decreased and the number of cells in G1 increased and decreased in S phase in L-periaxin gene knock-out RSC96 cell by flow cytometry and MTT assay.

Nanog influences the proliferative ability of HepG2 cells through the modulation of the cell cycle related proteins / 实用肿瘤学杂志

Objective To study the effect of pluripotency factor Nanog on the expression of the cell cy-cle related proteins,and then to explore its effect on the proliferative ability of HepG 2 cells.Methods TALENs gene editing tool was employed to induce mutation and downregulation expression of Nanog .T7 endonuclease 1 and genomic sequencing was used to analyze the mutation efficiency of Nanog .RT-PCR and western blot were used to determine the expression of mRNA and protein of Nanog ,respectively .Real-time cell based assay system was used to measure the proliferative ability of wild -type HepG2 cells and monoclonal HepG 2 cells with Nanog mutation.Results TALENs successfully induced mutation of Nanog gene .The targeting efficiency of mixed cells was analyzed by T7 endonuclease 1 approached 40%after two transfection with plasmid of Nanog -TALENs.Ad-ditionally,the Nanog mRNA expression level of monoclonal HepG 2 with Nanog mutation was downregulated by 3.4 times compared to the wild type HepG 2 cells,and the Nanog protein expression level was downregulated by 3.6 times.The cell cycle related proteins CyclinD1/D3,CyclinE1 and CDK2 expression were downregulated in monoclonal HepG2 with Nanog mutation in comparison to the wild type HepG 2 cells.Conclusion Nanog plays a role in influencing the proliferative ability of HepG 2 cells through modulating the expression of the cell cycle re-lated proteins CyclinD1/D3,CyclinE1 and CDK2.The downregulation expression of Nanog can inhibit the prolif-erative capacity of HepG 2 cells via the regulation of the cell cycle related proteins .

Functional disruption of human leukocyte antigen II in human embryonic stem cell

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allo-graft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA(-/-)hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA(-/-)hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA(-/-)hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA(-/-)hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA(-/-)hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4+ T cells, which are the main effector cells of cellular immunity in allograft.