جستجوی مقالات مرتبط با کلیدواژه « cho cell » در نشریات گروه « پزشکی »

CRISPR/Cas9 gene editing technology has revolutionized gene manipulation by providing the opportunity of gene knock out/in, transcriptional modification and base editing. The application of this system extended into different eras of biology, from cell development to animal modeling. Various generations of CRISPR technology have been developed to make genome editing easy which resulted in rapid protocols for amelioration of a large genome.

we established a simple protocol for gene manipulation in Chinese hamster ovary (CHO) cells to achieve a Caspase 7 deficient cell line by using combination of all-in-one CRISPR technology and CRISPR/ Cas9 homology-independent targeted integration (CRISPR HITI).

the findings of this study indicated that using CRISPR knocking in/out technology facilitates genomic manipulation in CHO cells. Integration of EGFP in target locus of caspase 7 gene made the selection of knockout CHO cell line easy which achieved by cell sorting and single-cell cloning.

this system introduces an effective targeting strategy for multiplex genome engineering, coinciding gene integration which simplified the selection of desired genomic characteristics.

Toxoplasmosis is a common infection all around the world. During pregnancy; it may lead to congenital disorders or abortion in human and animals. Severe damage of toxoplasmosis indicates to require effective vaccine. One of dense granules antigen is GRA4 that secrete from tachyzoite and bradyzoite. GRA4 genome is unique without intron and is one of the major immunogenic proteins from Toxoplasma gondii. We confirmed the cloning of GRA4 gene into pcDNA3 by restriction enzyme and PCR of GRA4 gene with pcGRA4 plasmids as template. Then with using calcium- phosphate method we transfected the pcGRA4 into CHO (Chinesehamster ovary) cells. The yielded protein was separated by SDS-PAGE and moved by electroblotting to nitrocellulose paper. Result of SDS-PAGE analysis showed the appearance of band approximately 42 kDa which was absent in the negative control, that was able to identify toxoplasmosis antibody IgM+ serum in western blot analysis. pcGRA4 plasmid is able to synthesis of antigenic protein in CHO cells. The ability of pcGRA4 for induction of protective immune response against toxoplasmosis will be evaluated in mouse model.

Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons.

Toxoplasmosis is an opportunistic protozoan infection with a high prevalence in a broad range of hosts infecting up to one-third of the world human population. Toxoplasmosis leads to serious medical problems in immunocompromised individuals and fetuses and also induces abortion and mortality in domestic animals. Therefore, there is a huge demand for the development of an effective vaccine. Surface Antigen 1 (SAG1) is one of the important immunodominant surface antigens of Toxoplasma gondii, which interacts with host cells and primarily involved in adhesion, invasion and stimulation of host immune response. Surface antigen 1 is considered as the leading candidate for development of an effective vaccine against toxoplasmosis. The purpose of this study was to clone the major surface antigen1 gene (SAG1) from the genotype 1 of T. gondii, RH strain into the eukaryotic expression vector pVAX1 in order to use for a DNA vaccine. Genomic DNA was extracted from tachyzoite of the parasite using the QIAamp DNA mini kit. After designing the specific primers, SAG1 gene was amplified by Polymerase Chain Reaction (PCR). The purified PCR products were then cloned into a pPrime plasmid vector. The aforementioned product was subcloned into the pVAX1 eukaryotic expression vector. The recombinant pVAX1-SAG1 was then transfected into Chinese Hamster Ovary (CHO) cells and expression of SAG1 antigen was evaluated using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), Immunofluorescence Assay (IFA) and Western Blotting (WB).: The cloning and subcloning products (pPrime-SAG1 and pVAX1-SAG1 plasmid vectors) of SAG1 gene were verified and confirmed by enzyme digestion and sequencing. A 30 kDa recombinant protein was expressed in CHO cells as shown by IFA and WB methods. The pVAX1 expression vector and CHO cells are a suitable system for high-level recombinant protein production for SAG1 gene from T. gondii parasites and are promising approaches for antigen preparation in vaccine development.

Proteinase 3(PR3) is a human polymorphonuclear leukocyte serine proteinase and is the main target antigen for antineutrophil cytoplasmic antibodies (ANCA) found in Wegener's granulomatosis (WG). We developed a stable expression system for conformationally intact recombinant PR3 (rPR3) in Chinese hamster ovary cells (CHO-cells). The part of PR3 cDNA that encoded the active form of PR3 was selected by using appropriate primers, and a signal sequence was also added in front ofPR3 eDNA. The signal sequencePR3 (S-PR3) was cloned into the pME 18 expression vector and the result product was electroporated into E. coli (DH5 a strain). After isolation and purification, the presence of pMEI8-S-PR3 was confirmed by using appropriate restriction endonuclease and agarose gel electrophoresis. The pMEI8- S-PR3 was electroporated with CHO-cells and the presence of rPR3 was tested in culture medium after 10 days. There was 12 ng/mL rPR3 in culture medium that had activity and was recognized by ANCA in ELISA.