Construction of Human Neuromuscular Disease-Related Gene Site-Specific Mutant Cell Line by Cas9 Mutation System

Objective: to construct human neuromuscular disease-related gene site-specific mutant cell line by Cas9 mutation system. Methods: according to the principle of CRISPR/Cas9 target design, the exon region of CXCR4 gene sequence was found in the National Center for Biotechnology Information (NCBI) of the United States. Two sgRNAs were designed. Lenticrisprv2 was used as the vector to construct the lenticrisprv2-sgrna recombinant plasmid, which was transformed into the sensitive stbl3 strain. The monoclonal sequencing was selected to verify and expand the culture of the plasmid, then it was transferred to 293T cells for packaging to a slow virus. The virus was collected and infected with 4T1 cells. The monoclonal cells were isolated and cultured by puromycin screening and limited dilution method. The genomic DNA of the selected monoclonal cells was extracted and the DNA fragment near the knockout site was amplified by PCR and sequenced. Results: one cell line had 6 deletion mutations, including DYSF mutation site of neuromuscular disease gene and HEK293T cell model knocked out by DYSF mutation site of neuromuscular disease gene. Conclusion: the recombinant plasmid targeting CXCR4 gene was obtained by CRISPR/Cas9 system, and the human neuromuscular disease-related gene site-specific mutant cell line was successfully constructed.

Construction of New Genetically Engineered Vaccine Strain for O-type Foot-and-Mouth Disease of Pig

Foot-and-mouth disease is an acute and highly contagious viral infectious disease. Although the foot and mouthdisease vaccine has been applied in some parts of the world since the beginning of the 20th century, the currentepidemic of foot and mouth disease in the world is still serious and constitutes an obstacle to the trade of animal andanimal products in the world. The porcine pseudorabies virus gene deletion strain PRV TK-/ gE-/ LacZ +, which isconstructed by our laboratory, has the advantages of good safety, large capacity and high recombination effi ciency. Inthis study, the artifi cial O-type foot-and-mouth disease P1 gene as an antigen gene, UbiP1 fused with ubiquitin (Ub)as another antigen gene to enhance cellular immunity, and then shRNA designed for FMD 3B gene and porcine IFN-[gamma] with antiviral and immune-regulatory eff ects. The two functional genes were constructed in turn to constructa transfer vector with four functional genes. The transfer vector was transfected into TK-/ gE- / LacZ+ cells with PRVdeletion vector, and the plasmids were purifi ed and identifi ed. The recombinant pseudorabies virus, which containsthe six functional genes of P1 gene, UbiP1, shRNA and IFN-γ, was obtained, which laid the foundation for furtherconstruction of new genetically engineered vaccine.

Study on the Coniferous Characters of Pinus yunnanensis and Its Clustering Analysis

Pine is a relatively easy genus for intermediate hybridization. It has been widely believed that there should be anatural hybrid population in the distribution of Pinus massoniona Lamb. and Pinus hangshuanensis Hsia, that is, theexcessive type of external form between Pinus massoniana and Pinus taiwanensis exist. This paper mainly discussesthe traits and clustering analysis of coniferous lozeng in Huangshan scenic area. This study will provide a theoreticalbasis for the classification of long and outstanding Huangshan Song and so on. At the same time, it will providereference for the phenomenon of gene seepage between the two species.

Research Progress and Application Prospect of IPS Cells

Differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPS cells) by introducingspecific transcription factors. This technique avoids immune rejection and ethical problems in stem cell research.A great revolution in the fi eld of science. As with embryonic stem cells (ES cells), iPS cells are able to self-renewand maintain undiff erentiated state. In vitro, iPS cells can be induced to diff erentiate into a variety of mature cells,therefore, iPS cells in theoretical research and clinical applications are extremely valuable. IPS cell diff erentiationand transplantation in the treatment of blood diseases have a great use, iPS cells can treat nervous system diseases,to provide in vitro disease model, to study the mechanism of disease formation, screening new drugs and thedevelopment of new to provide a new treatment The The use of iPS cells as a nuclear donor cell, with the appropriatereceptor cells after fusion can be directly obtained transgenic animals. Not only can improve the genetic nature ofanimals, but also can break the boundaries of species and get the new animal traits that cannot achieve by usingtraditional mating methods. The research of iPS cells has been widely concerned, and it is the research hotspot in cellbiology and molecular biology. In this paper, the defi nition of iPS cells, the acquisition of iPS cells, the history ofdevelopment, the signifi cance of research, the progress of research, the application of iPS cells, and the problems ofiPS cells were reviewed.

A Preliminary Study on the Relationship between Micronucleus Formation and Cell Cycle

Micronucleus is an abnormal structure in eukaryotic cells. It is the result of various physical and chemical factors,such as radiation and chemical drugs acting on dividing cells. It is generally believed that micronuclei are originatedin backward chromosomes and fragments that have lost centromere and are formed at the end of mitosis. However,other experiments suggest that interphase cells can also form micronuclei. In this study, human peripheral bloodlymphocytes were cultured in vitro and cyclophosphamide 20 ug/ml was injected at specifi c times of the cell cycle toobserve the relationship between micronuclei formation and cell cycle. The experimental results confi rmed that thecell cycle micronucleus formation.

Plasma Mutation Breeding of High Yield γ-Aminobutyric Acid Lactic Acid Bacteria

γ-amino butyric acid (GABA) is a non-protein amino acid widely distributed in nature. It has analgesic effect,promotes growth hormone secretion and prevents the physiological function of Alzheimer's disease.In order toimprove the yield of GABA, the strain of strain HX-3-6 was tested by Atmospheric and Room Temperature Plasma(ARTP), and the strain with more than 90% lethality was selected for fermentation, and through the gradient plateaccording to the level of GABA concentration and colony size of the screening. Then, use the fermentation mediumfor re-screening and genetic stability test. The yield of the mutant strain L-120-1 was 5.828 g/L, which was 18.84%higher than that before the mutation (4.904). The mutant strain L-120-1 was used as the starting strain for ARTPmutagenesis. The yield of the stable mutant strain 90s-high was 6.178g / L, which was 16.15% higher than that beforethe mutation (5.319g / L). The yield of HX-3-6 strain was higher than that of GABA mutant strain 90s-high 2, whichwas stable and high yield GABA was 25.98%.