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10.1038/s41596-021-00491-8 http://scihub22266oqcxt.onion/10.1038/s41596-021-00491-8 33514944!8168523!33514944     free     free     free Deprecated: Implicit conversion from float 235.6 to int loses precision in C:\Inetpub\vhosts\kidney.de\httpdocs\pget.php on line 534       Nat+Protoc 2021 ; 16 (3): 1761-1784 Nephropedia Template TP {{tp|p=33514944|t=2021. Engineering SARS-CoV-2 using a reverse genetic system |pdf=|usr=}}{{33514944}} gab.com Text Engineering SARS-CoV-2 using a reverse genetic system JO: Nat Protoc http://www.kidney.de/pget.php?&tw=1&pmid=33514944 #FOAvip Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research. Twit Text FOAVip Engineering SARS-CoV-2 using a reverse genetic system ????Nat Protoc http://www.kidney.de/pget.php?&tw=1&pmid=33514944 #FOAvip Twit Text # Free Paper on # # # # # LINK http://www.kidney.de/pget.php?&tw=1&pmid=33514944 #FOAvip English Wikipedia <ref>{{Cite pmid|33514944}}</ref> Engineering SARS-CoV-2 using a reverse genetic system #MMPMID33514944 Xie X; Lokugamage KG; Zhang X; Vu MN; Muruato AE; Menachery VD; Shi PY Nat Protoc 2021[Mar]; 16 (3): 1761-1784 PMID33514944show ga Reverse genetic systems are a critical tool for studying viruses and identifying countermeasures. In response to the ongoing COVID-19 pandemic, we recently developed an infectious complementary DNA (cDNA) clone for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse genetic system can be used to rapidly engineer viruses with desired mutations to study the virus in vitro and in vivo. Viruses can also be designed for live-attenuated vaccine development and engineered with reporter genes to facilitate serodiagnosis, vaccine evaluation and antiviral screening. Thus, the reverse genetic system of SARS-CoV-2 will be widely used for both basic and translational research. However, due to the large size of the coronavirus genome (~30,000 nucleotides long) and several toxic genomic elements, manipulation of the reverse genetic system of SARS-COV-2 is not a trivial task and requires sophisticated methods. Here, we describe the technical details of how to engineer recombinant SARS-CoV-2. Overall, the process includes six steps: (i) prepare seven plasmids containing SARS-CoV-2 cDNA fragment(s), (ii) prepare high-quality DNA fragments through restriction enzyme digestion of the seven plasmids, (iii) assemble the seven cDNA fragments into a genome-length cDNA, (iv) in vitro transcribe RNA from the genome-length cDNA, (iv) electroporate the genome-length RNA into cells to recover recombinant viruses and (vi) characterize the rescued viruses. This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research. |DNA, Viral/genetics[MESH] |Genetic Engineering/*methods[MESH] |Genome, Viral/genetics[MESH] |Reverse Genetics/*methods[MESH]Engineering SARS-CoV-2 using a reverse genetic system (epmc).pdf DeepDyve Pubget Overpricing