Construction and Packaging of CaMKIIγ Lentiviral Vector

Xibing Zhang; Fang Liu; Ruichao Wu; Yang Gao; Jianghua Ran

doi:10.47297/taposatWSP2633-456915.20220303

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Construction and Packaging of CaMKIIγ Lentiviral Vector

Volume 3, Issue 3

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Construction and Packaging of CaMKIIγ Lentiviral Vector
Construction and Packaging of CaMKIIγ Lentiviral Vector
The Affiliated Calmette Hospital of Kunming Medical University
Vol. 3, Issue 3, Pages: 75-80(2022)
Published： 2022 ，
DOI：10.47297/taposatWSP2633-456915.20220303
Accepted：

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XIBING ZHANG, FANG LIU, RUICHAO WU, YANG GAO & JIANGHUA RAN. (2022). Construction and Packaging of CaMKIIγ Lentiviral Vector. Theory and Practice of Science and Technology, 3(3), 75-80..
DOI：

XIBING ZHANG, FANG LIU, RUICHAO WU, YANG GAO & JIANGHUA RAN. (2022). Construction and Packaging of CaMKIIγ Lentiviral Vector. Theory and Practice of Science and Technology, 3(3), 75-80.. DOI： 10.47297/taposatWSP2633-456915.20220303.

摘要

Abstract

To construct

identify as well as package the lentiviral silence and overexpression vector of calmodulin dependent protein kinase IIγ (CaMKIIγ). Construct the assembled lentiviral vector of LVRU6MP[shRNA]mcherry:SV40:Puro-U6 and LV201[CaMKIIγ]eGFP:SV40:Puro-IRES. The blank control group

NC group

silence group

and overexpression group were set

and the lentivirus was packaged. The titer of the packaged virus was determined by qPCR absolute quantitative method. The sequencing results showed that the target gene sequence was completely consistent with the CaMKIIγ gene sequence published by GenBank. 48h after transfection of the target plasmids LVRU6MPNC

sh-CaMKIIγ and OE-CaMKIIγ

more than 90% of 293Ta cells expressed red fluorescence and green fluorescence

respectively. The titers of packaged viruses in each group were determined by qPCR absolute quantification

which were all greater than 1×108copies/ml. We successfully constructed the lentivirus silence and overexpression vector of the CaMKIIγ gene

and the lentivirus was successfully packaged and titered

laying a solid foundation for the follow-up study of the CaMKIIγ gene.

关键词

Keywords

CaMKIIγLentiviralGene silenceGene overexpression

references

Khomich O, Ivanov AV, Bartosch B. “Metabolic Hallmarks of Hepatic Stellate Cells in Liver Fibrosis”[J].Cells,2020,9(1): 24.[2] Ping An,Yihao Tian,Mingkai Chen,et al. “Ca(2+)/calmodulin-dependent protein kinase II mediates transforming growth factor-β-induced hepatic stellate cells proliferation but not in collagen α1(I) production”[J].Hepatol Res,2012,42(8):806-18.[3] Pepke S,Kinzer-Ursem T,Mihalas S,et al. “A dynamic model of interactions of Ca2+,calmodulin,and catalytic subunits of Ca2+/calmodulin-dependent protein kinase II”[J]. PLoS Comput Biol,2010,6(2): e1000675.[4] Liu H,Wang L,Dai L,et al. “CaMK II/Ca2+ dependent endoplasmic reticulum stress mediates apoptosis of hepatic stellate cells stimulated by transforming growth factor beta 1”[J].Int J Biol Macromol,2021,172: 321-29. [5] Timmins JM,Ozcan L,Seimon TA,et al. “Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways”[J]. J CLIN INVEST,2009,119(10):2925-41.

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