ISOLATION AND IDENTIFICATION OF THE RELATED GENE MEDIATING γ-RADIATION-INDUCED APOPTOSIS IN HUMAN HL- 60 CELLS

To identify the member of the caspase family proteases involved in γ radiation induced apoptosis in HL 60 cells, using degenerated oligonucleotide primers encoding the highly conserved peptides, which were present in all known caspases, RT PCR was performed on poly (A) RNA from the γ radiation induced apoptotic HL 60 cells. Then, cloned and sequenced to identify the amplified DNA fragments. The results showed that the amplified DNA fragments were identified with a part of caspase 3 cDNA. It indicated that caspase 3 was involved in γ radiation induced apoptosis in HL 60 cells and may be the pivotal element of radiation induced apoptosis.

SEQUENCE STUDY AND RETROVIRUS MEDIATED GENE TRANSFER IN MAMMALIAN CELL SYSTEMS OF HUMAN INTERLEUKIN 2 cDNA

Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.

Cell mediated gene therapy: A guide for doctors in the clinic

The recent approval of gene therapy products in Europe and Asia and the upsurge of gene therapy products in clinical trials signal the rebound of this technology not only for many orphan diseases but also for non-life threatening diseases. Following the success of induced pluripotent stem （iPS） cells in research, other modifed ex vivo gene therapies are also knocking on the door of the clinic. Historically, gene therapy has experienced many ups and downs and still faces many challenges.During the past 10 years, many new ideas have been tried, and the goal of making this technology a more effective treatment modality through greater safety and control is coming within reach. The frst clinical trial of iPS cells has begun, and cell mediated gene therapy products have reached phase Ⅲ in some countries. The potential for tumorigenicity and immunogenicity are still concerns with these products, so physicians should understand the biological aspects of engineered cells in the clinic. In this review article, we attempted to provide a summary update of the current state of knowledge regarding this technology： that is, we reviewed products that have fnished clinical trials, are still in clinical trials and/or are at the research stage. We also focused on the challenges, future directions, and strategies for making this technology available in the clinic. In addition, the available measures for making gene therapy products safer are within the scope of this article. It is also important to understand the manufacturing process for gene therapy products, because cell characteristics can change during the cell expansion process. When physicians use gene therapy products in the clinic, they should be aware of the viability, temperature sensitivity and stability of these cells because biologic products are different from chemical products. Although we may not be able to answer all possible questions and concerns, we believe that this is the right time for physicians to increase their interest in and understanding of this evolving technology.

Studies on Introduction of Arrowhead Proteinase Inhibitor Gene into N． tobacco Protoplasts

The Arrowhead Proteinase Inhibitor(API)gene was introduced into the protoplasts or mesophyll cells of N.tobacco by PEG-mediated.The transformed protoplasts undergoing dirrerentiation of callus and regeneration of plantlet have been growing into transgenic plants. Restriction endonuclease analysis of products amplificated by PCR indicates the existence of the API gene in the transformed plantlet.The extract of the leaves from the transformed plants shows trypsin inhibitory activity,which indicates the expression of the introduced API gene and the transformed plants can accumulate the inhibitor. However, the variation of the inhlbitory activity of the transformed plants reveals the importance of the integration site of the API gene in the genome.

Characterization and target gene analysis of microRNAs in the antennae of the parasitoid wasp Microplitis mediator

MicroRNAs(miRNAs),a class of non-coding single-strand RNA molecules encoded by endogenous genes,are about 21–24 nucleotides long and are involved in the post-transcriptional regulation of gene expression in plants and animals.Generally,the types and quantities of miRNAs in the different tissues of an organism are diverse,and these divergences may be related to their specific functions.Here we have identified 296 known miRNAs and 46 novel miRNAs in the antennae of the parasitoid wasp Microplitis mediator by high-throughput sequencing.Thirty-three miRNAs were predicted to target olfactory-associated genes,including odorant binding proteins(OBPs),chemosensory proteins,odorant receptors(ORs),ionotropic receptors(IRs)and gustatory receptors.Among these,17 miRNAs were significantly highly expressed in the antennae,four miRNAs were highly expressed both in the antennae and head or wings,while the remaining 12 miRNAs were mainly expressed in the head,thorax,abdomen,legs and wings.Notably,miR-9a-5p and miR-2525-3p were highly expressed in male antennae,whereas miR-1000-5p and novel-miR-13 were enriched in female antennae.The 17 miRNAs highly expressed in antennae are likely to be associated with olfaction,and were predicted to target one OBP(targeted by miR-3751-3p),one IR(targeted by miR-7-5p)and 14 ORs(targeted by 15 miRNAs including miR-6-3p,miR-9a-5p,miR-9b-5p,miR-29-5p,miR-71-5p,miR-275-3p,miR-1000-5p,miR-1000-3p,miR-2525-3p,miR-6012-3p,miR-9719-3p,novel-miR-10,novel-miR-13,novel-miR-14 and novel-miR-28).These candidate olfactory-associated miRNAs are all likely to be involved in chemoreception through the regulation of chemosensory gene expression in the antennae of M.mediator.

ZFN,TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis:A disconnect between somatic and germinal cells

Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break

Efficient Gene Targeting in Zebrafish Mediated by a Zebrafish-Codon-Optimized Cas9 and Evaluation of Off-Targeting Effect

Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human cells as well as in most model organisms, including zebrafish （Danio rerio）, mouse, and fruit fly （Chang et al., 2013; Cong et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jao et al., 2013; Shen et al., 2013; Wei et al., 2013）. Its application in zebrafish is particu- larly attractive due to the ease of handling this organism and the simple application of this method by direct injection of Cas9/ gRNA. However, the information about its specificity in this organism is very limited and needs further evaluation. In addition, it is conceivable that a Cas9 mRNA optimized for zebrafish codon preference could enhance its activity.

ADENOVIRUS MEDIATED GENE TRANSFER OF VASCULAR SMOOTH MUSCLE CELLS AND ENDOTHELIAL CELLS IN VITRO

Introducing foreign gene(s) into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is the pre-require-ment of gene therapy for cardiovascular diseases. We have explored the use of adenoviral vectors (Adv-CMV / LacZ) to transfer LacZ gene into cultured VSMCs and ECs. Our results demonstrated that adenoviral vectors transfered foreign gene into VSMCs and ECs high-efficiently with dose-dependent response pattern. The frequencies of transfection reached 100% at the viral titer of 109 pfu / ml. Comparing the sensitivities of VSMCs and ECs to adenoviral vectors, we found that ECs were more sensitive than VSMCs, of which the frequencies of transfection in ECs reached 80% while in VSMCs only 40% for 8 hrs after transfection. In addition, the transfection of ECs and VSMCs with adenoviral vectors was partly blocked by monoclonal antibodies to Fiber and Core protein of the adenoviral capsid, but not by monoclonal antibody to Hcxon protein. It is suggested transfection of ECs and VSMCs with adenovirus vectors is mediated by Fiber or Core protein of adenoviral capsid proteins.

CRISPR/Cas9 mediated gene editing helps pig fight cold and fat

Supported by the National Natural Science Foundation of China,a research team led by Prof.Zhao Jianguo(赵建国)and Jin Wanzhu(金万洙)from the State Key Laboratory of Stem Cell and Reproductive Biology,Institute of Zoology,Chinese Academy of Sciences and Prof.Wang Yanfang(王彦芳)from

IN VIVO SOMATIC GENE TRANSFER WITH IkB AND TRUNCATED JUN DEPENDENCE OF TNF MEDIATED INTRAVASCULAR FIBRIN FORMATION ON NFkB AND AP-1

Recently it has been shown that induction of tissue factor (TF) by TNF in vitro is dependent on a concerted action of NFkB and AP-1. However it remains unknown, if TF can mediate intravascular fibrin formation and if NFkB and AP-1 are involved in intravascular fibrin formation in vivo. When mice with Meth-A sarcomas were injected with TNF; TF was expressed by vascular endothelium of the tumor,