Recombinant adenovirus has been used extensively to express foreign genes because of its high infection efficiency and its ability to infect a broad spectrum of cell-types. In this review, we describe these advantages and how it works by using a recombinant adenovirus to study the NS2 of Hepatitis C virus (HCV) and focal adhesion kinase(FAK)- related non-kinase(FRNK). Taken the research we have done together recombinant adenovirus demonstrates a very useful vector for transferring genes of interest from the outside

Cao Jun

Chaohong Liu

Xinwen Chen

English

Recombinant adenovirus has been used extensively to express foreign genes because of its high infection efficiency and its ability to infect a broad spectrum of cell-types. In this review, we describe these advantages and how it works by using a recombinant adenovirus to study the NS2 of Hepatitis C virus (HCV) and focal adhesion kinase(FAK)- related non-kinase(FRNK). Taken the research we have done together recombinant adenovirus demonstrates a very useful vector for transferring genes of interest from the outside.Review | Biohelikon : Cell Biolog

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1Biohelikon: Cell Biology, 2013 1:3Review ArticleRecombinant adenovirus, apowerful vector to transfer geneChaohong Liu1*, Cao Jun2, Xinwen Chen31 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA2 Department of Gastroenterology, People's Hospital of Wuhan University, Wuhan 430060, China3 State key laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, China* Corresponding author, Email: cliu1234@umd.edu AbstractRecombinant adenovirus has been used extensively to express foreign genes because of its high infectioneﬃciency and its ability to infect a broad spectrum of cell-types. In this review, we describe these advantagesand how it works by using a recombinant adenovirus to study the NS2 of Hepatitis C virus (HCV) and focaladhesion kinase(FAK)- related non-kinase(FRNK). Taken the research we have done together recombinantadenovirus demonstrates a very useful vector for transferring genes of interest from the outside.KeywordsRecombinant adenovirus, NS2, FRNK Recombinant adenovirus has been used extensivelyfor transferring foreign genes because of severalunique advantages. For example, it can infect mosttypes of cells. High titer of virus and high expressionlevel of foreign genes were easy to be obtained[1] . The most commonly used adenovirus is serotype5 human adenovirus with a linear and doublestranded DNA genome [1] . Currently the engineeredadenoviral vectors have made the expression offoreign genes much more convenient as comparedto the previous adenoviral vectors. The most criticalimprovement is that the recombination processhappens in E.coli instead of mammalian cells, whichhave low recombination eﬃciency and needs plaquepuriﬁcation [1] . The engineered adenoviral vectorsinclude a GFP marker that can easy identify theeﬃciency of transfection and infection. These uniqueadvantages of newly engineered adenovirus havebeen applied to most of our previous research.An important non-structural gene of HCV genomeis ns2. NS2 was shown to inhibit several promoters inhepatic and nonhepatic cell lines, such as promotersthat control the expression of CMV and SV40 [2] .NS2can block apoptosis by inhibiting the release ofcytochrome C [3] . In order to study the function ofNS2 in the HCV life cycle, a system that can express ahigh level of NS2 in hepatic cell lines, such as HepG2,would be needed. From our previous experience,the level of expression of foreign genes in HepG2cells is usually low with traditional methods, suchas lipofectin transfection. Instead we constructed arecombinant adenovirus that can express ns2 with aGFP marker. The titer of this recombinant adenoviruscan be high up to 109 PFU/ml after propagation in 293cells [4] . The infection eﬃciency of this recombinantadenovirus in HepG2 cells can be up to 90% [4] .This high infection eﬃciency contributes to the highexpression of ns2 in HepG2 cells [4] . The GFP markercarried by recombinant adenovirus also make it easyto observe the antiviral eﬀect of natural productsuch as dioscin [5] .FAK (focal adhesion kinase), also referred toas cell adhesion kinase (CAK)-Β, related adhesionfocal tyrosine kinase (RAFTK) or calcium-dependentprotein tyrosine kinase (CADTK) [6] , belongs tothe members of the FAK family of non-receptorprotein tyrosine kinases. FAK has been shown tobe closely related with cell adhesion, migrationand growth regulation. Increased FAK expressionhas been correlated with increased cancer cellmotility, invasiveness and proliferation [6] . FRNK islocated at the C-terminal region of FAK. It doesn'thave kinase activity or the autophosphorylation siteTyr-397 that is seen in FAK. FRNK is a dominant-negative inhibitor of FAK by competitive inhibition[7] . Recently, Cao et al., not only constructeda recombinant adenovirus that expresses murineFRNK [8] that can inhibit tumor cell motility andinvasion but also a recombinant adenovirus thatexpresses human FRNK(hFRNK) [9] . The titer ofthese two recombinant adenovirus can be highup to 1012 PFU/ml, which facilitates the followingresearch on the study of the function of FRNK.Colo320, a colonic carcinoma cell line, is diﬃcult toundergo transfection with lipofectin. Cao et al., useda constructed adenovirus that expresses FRNK toinfect the colo320 and studied the eﬀect of FRNK onthe phosphorylated 190RhoGAP and RhoA Activityin colo320WT colorectal cells. Cao et al., foundthat hFRNK can inhibit expression of phosphorylatedp190RhoGAP and enhance RhoA activity in the cellsstimulated with Gastrin17 [10] , which activatesbeta-catenin/Tcf-4 signaling in Colo320WT cells,thereby leading to an over-expression of c-myc andcyclin D1. This mechanism probably works withhFRNK blocking FAK phosphorylation and the FAK2pathway [11] . Cao et al., also found that adenoviruscarrying hFRNK can inhibit abnormal distributionof E-cadherin and beta-catenin in the gastrin17-stimulated cells. The mechanism probably workssimilarly with hFRNK dephosphorylating FAK andblocking the FAK pathway [12] .Generally, recombinant adenovirus is an eﬃcientsystem that is characterized by high eﬃciencyinfection and high expression level of foreign genes.Furthermore, the important and critical advantage isthat the high eﬃciency infection is cell independent,which enables us to study the eﬀect of interestedgenes in most of type of cells without the previousconstraint. The high expression level of foreign genesalso can allow us to save cost on the carrier thattransfers the foreign DNA into cells such as lipofectin.AcknowledgementWe thank Liu Lisa for critically reading themanuscript. This work was supported by a grant fromthe health department of Hubei province (JX1B027)of China. References1. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW,  et al.: A simpliﬁedsystem for generating recombinant adenoviruses. Proc Natl AcadSci U S A 1998, 95: 2509-25142. Dumoulin FL, von dem Bussche A, Li J, Khamzina L, Wands JR,et al.: Hepatitis C virus NS2 protein inhibits gene expression fromdiﬀerent cellular and viral promoters in hepatic and nonhepatic celllines. Virology 2003, 305: 260-2663. Erdtmann L, Franck N, Lerat H, Le Seyec J, Gilot D,  et al.: Thehepatitis C virus NS2 protein is an inhibitor of CIDE-B-inducedapoptosis. J Biol Chem 2003, 278: 18256-182644. Chao-hong Liu, Xin-wen Chen:Cloning of HCV ns2 Gene and itsExpression in Prokaryotic and Mammalian cells. Virologica Sinica2006, 21: 217-2205. Liu C, Wang Y, Wu C, Pei R, Song J,  et al.: Dioscin's antiviral eﬀect invitro. Virus Res 2013, 172: 9-146. J T Parsons:Focal adhesion kinase: the ﬁrst ten years. J Cell Sci 2003,116: 1409-14167. K Nolan, J Lacoste, J T Parsons:Regulated expression of focaladhesion kinase-related nonkinase, the autonomously expressed C-terminal domain of focal adhesion kinase. Mol Cell Biol 1999, 19:6120-61298. J Cao, D MingYou , C Yin, L ChaoHong , Y JiePin,  et al.:Constructionof Inhibiting Tumor Cell Motility and Invasion Murine FRNKRecombinant adenovirus Vector. Chinese Journal of ExperimentalSurgery 2006, 23: 1502-15049. C Jun, L Chaohong, L Qisheng, Q Yuanling, Y Honggang:Construction of Human FRNK Recombinant Adenovirus Vector.Medical Journal of Wuhan University 2007, 28: 265-26910.J Cao, Y Jie-ping, L Chao-hong, C Xin-wen, L He-sheng:Eﬀectsof adenovirus vector expression hFRNK on phosphorylated190RhoGAP and RhoA Activity in colo320WT colorectal cells.Chinese Journal of Practical Internal Medicine 2007, 27: 581-58411.J Cao, JP Yu, CH Liu, L Zhou, HG Yu:Eﬀects of gastrin 17 on beta-catenin/Tcf-4 pathway in Colo320WT colon cancer cells. World JGastroenterol 2006, 12: 7482-748712.J Cao, JP Yu, CH Liu, XW Chen, S Liu, et al.: Eﬀects of hFRNK onE-cadherin/beta-catenin in colon cancer cells in vitro, ZhonghuaZhong Liu Za Zhi 2007, 29: 346-350

Recombinant adenovirus, a powerful vector to transfer gene

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Recombinant adenovirus, a powerful vector to transfer gene

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