ABSTRACT
ABSTRACT Are presented results of experimental pig kidney xenotransplantation in Brazil, which aims to reduce the waiting list mortality due to shortage of organs. Recent clinical results obtained abroad are commented.
RESUMO Apresentam-se resultados de xenotransplante suíno de rim experimental no Brasil que visa reduzir as listas de espera nas quais falecem muitos inscritos à espera do transplante. Comentam-se os recentes resultados clínicos obtidos no exterior.
Subject(s)
Animals , Kidney Transplantation , Swine , Transplantation, Heterologous/methods , Brazil , Waiting Lists , KidneyABSTRACT
RESUMEN La tecnología CRISPR/Cas es un método simple, rápido y extremadamente eficiente para la edición de genes. Con el objetivo de describir los principios y aplicación médica de dicha tecnología, se realizó una revisión bibliográfica en Pubmed, SciELO, Google académico y Cochrane Library, con los descriptores "edición de genes", "edición del genoma", "sistemas CRISPR-Cas" y "proteína 9 asociada a CRISPR". El sistema CRISPR/Cas9 comprende una endonucleasa Cas y dos tipos de RNA. Cas corta el DNA del fago invasor en segmentos, los cuales se integran a la secuencia CRISPR como espaciadores. Posteriormente, la secuencia CRISPR se transcribe para generar crRNA y tracrRNA, que forman una estructura de RNA de doble hélice que recluta Cas para el clivaje. La introducción del sistema al interior celular se produce con plásmidos, RNA o ribonucleoproteínas. Una secuencia de localización nuclear permite que CRISPR/Cas9 entre al núcleo. La tecnología CRISPR/Cas9 es una eficiente herramienta para la edición precisa de genes con gran impacto en la investigación científica.
ABSTRACT CRISPR/Cas technology is a simple, fast and extremely efficient method for gene editing. In order to describe the principles and medical application of this technology, a bibliographic review was carried out in Pubmed, SciELO, academic Google and the Cochrane Library, with the descriptors "gene editing", "genome editing", "CRISPR-Cas systems", and "CRISPR-associated protein 9". The CRISPR/Cas9 system comprises a Cas endonuclease and two types of RNA. Cas cuts the invading phage DNA into segments, which are integrated into the CRISPR sequence as spacers. Subsequently, the CRISPR sequence is transcribed to generate crRNA and tracrRNA, which form a double-stranded RNA structure that recruits Cas for cleavage. The introduction of the system into the cell interior occurs with plasmids, RNA or ribonucleoproteins. A nuclear localization sequence allows CRISPR / Cas9 to enter the nucleus. CRISPR/Cas9 technology is an efficient tool for precise gene editing with great impact on scientific research.
ABSTRACT
BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.
Subject(s)
Animals , Cell Survival , Apoptosis , Cell Proliferation , Caspase 7/deficiency , Cricetulus , Cricetinae , CHO Cells , Caspase 7/geneticsABSTRACT
Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9),a cluster of regularly spaced short palindromic repeats,is a natural immune defense system for bacteria and archaea to identify themselves and exogenous invading DNA fragments,protecting them from viruses.In recent years,CRISPR/Cas9 has become a revolutionary gene editing tool.Its specific targeted spot-cutting ability also plays an important role in nucleic acid detection,bacterial typing,etc.,and has shown great application potential in the field of medical testing.Based on the latest researches,this paper reviews the progress of CRISPR/Cas9 application in the new techniques of nucleic acid detection,pathogen typing and bacterial evolution in laboratory medicine,and also summarizes the application prospect of CRISPR technology in the field of laboratory medicine.