Reverse genetics

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REVERSE GENETICS AND ITS APPLICATIONS : 

REVERSE GENETICS AND ITS APPLICATIONS By- Dr.Basavaraj Sajjanar IVRI

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Contents Definition Forward v/s Reverse Genetics Methods of RG RG of viruses Application Summary

What is reverse genetics? : 

What is reverse genetics? A methodology for studying the function of each gene in genomes Is a tool to investigate the impact of induced variation in a specific gene on the organism Rescue of virus entirely from transfecting cloned cDNA plasmids encoding the viral components positional cloning

Methods of reverse genetics : 

Methods of reverse genetics Three ways- 1. Large scale systematic mutagenesis 2.Random mutagenesis 3.Generation phenocopy mutant alleles

Normal (forward) genetics v/s Reverse genetics : 

Normal (forward) genetics v/s Reverse genetics

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To induce mutations in predefined genes of genome or To create null allele in the genome (gene knock out) GENE TARGETING Systematic gene targeting started recently in Saccharomyces cerevisiae Systematic gene deletion project – EUROFAN (European functional Analysis Network) in 1998 http:/mips.gsf.de/proj/eurofan/index http:/www.sequence.stansford.edu/group Large scale systematic mutagenesis

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PCR generated targeting cassets Marker gene between flanking sequence corresponding to the each yeast gene Target cassets replace gene from genome –h. recombination Select gene deleted strain of yeast by the help of marker Annotate the strain in the SGD database Subject each strain to many functional assay Determine function of missing gene method

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Gene targeting is achieved in higher organisms- mice, drosophila and in some plants Knock out organisms created for only few genes There is no systematic cataloging of knock out strains

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RG of Viruses A new field of study Rec .DNA tech enables…. Availability of viral genomes sequences Cloning of whole viral genomes Modification of viral genomes Generation of vectors,vaccines Chimeric viral genomes

DNA viruses : 

DNA viruses Poxviruses---- vaccinia virus, fowl pox, canary pox Herpesviruses----herpes simplex 1 virus Adenoviruses-------aviadenovirus as vectors Papovaviruses……papilloma virus Parvoviruses-----Adeno- associated viruses Are first viruses amenable to Rec. DNA tech Many DNA viruses subjected to RG study

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Recovery of genetically manipulated DNA viruses achived first time for SV- 40 by transfecting mutated cloned cDNA to produce defective mutant viruses (Goff and Berg ,cell,1976) Rescue of large DNA containing herpes virus utilizing h.recombination leading to construction of herpes virus variants(Post and Roizman,cell,1981) Live virus vector era began with development of vaccinia virus vector(Smith,Mackett and Moss, 1983)

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RG pox virus Construction of chimeric vaccinia virus-herpesvirus plasmids, rescue of rec. virus Vaccinia genomic DNA is digested with Hind ||| Clone it in the vector pBR-322to get pDP3 Obtain a TK gene segment with ends having Bam H | Clone it in Bam H | site of pDP3 Check the orientation and no copies of TK by digestion with Sst | and fragment analysis When these plasmids cotransfected with helper virus rec virus with TK gene generated Panicali and Paoleti ,pnas,1982

RNA viruses : 

RNA viruses Retroviruses -- Lentivirus Reoviruses-- Rotavirus Picornaviruses-- Poliovirus, FMD virus Flaviviruses---hepatitis C virus RG of RNA viruses depends on type of viral RNA genome Positive sense RNA Negative sense RNA

POSITIVE SENSE RNA VIRUSES : 

POSITIVE SENSE RNA VIRUSES Genome manipulation and rescue of positive sense RNA viruses is easier…….. Full length genomic RNA itself infectious No requirement of RNP Positive sense RNA acts as mRNA

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Cloned cDNA of poliovirus is infectious in mammalian cell Poliovirus- positive strand RNA genome of 7440 bps 3 cDNA copies cloned in 3 diff plasmids which together represent entire genome Three cDNA clones were joined forming single full length cDNA copy in Pst 1 site of pBR322 Transfection of rec. plasmid into CV-1or HeLa cell line produced infectious polio viruses (Raaniello and Baltimore,Science,1981)

Negative sense RNA viruses : 

Negative sense RNA viruses Non segmented Negative sense RNA viruses (NNSV) Segmented negative sense RNA viruses Paramyxoviridae--- sendai viruses, Rinder pest, human Para influenza viruses,3,measles, mumps, Newcastle disease Filoviridae------- Ebola ,Marburg viruses Rhabdoviridae ----- Rabies,VSV Bornaviridae--------- bornavirus

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Gene manipulation and rescue of negative sense RNA viruses was hindered for reasons like…… Genomic RNA of these viruses is not infectious it requires RNPcomplex Infectious particles deliver their own RNA dependent RNA polymerase to start virus replication

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In 1994 RG for Rabies virus entirely from cDNA was developed by Schnell,Mebatason and conzellman This method was quickly used to develop RG system for other NNSV like …… Human respiratory syncytial virus human parainfluenza virus NCD ,measels,VSV etc

PBRG of NNSV : 

PBRG of NNSV Transfect cell with plasmid encoding……… Full length genomic /antigenomic RNA Major proteins for transcription and replication Drive plasmid expression by phase T7 polymerase T7 polymerase made available by 1)cotransfection with plasmid encoding it 2)with helper vaccinia virus expressing it 3)using the cell lines which endogenously express it

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Recovery of infectious parainfluenza virus 3 from cDNA PI3-paramyxoviridae leniar,-ssRNA,15-16kbp NP,P,L associated with genome P,L –in replication ,transcription Transfect cultured cell with plasmid encoding genomic or antigenomic RNA Plasmid for major protein coding genes-NP,P,L Plasmid expression is driven by phage T7Polymerase

Segmented negative sense RNA viruses : 

Segmented negative sense RNA viruses Bunyamera virus (Bunyaviridae) produced entirely from cloned cDNA for three segments of vRNA (Bridgen and Elliot,pnas,1996) RG of influenza poses more challenges RNP complex------------- positive s mRNA viral RNA polymerase com Complexity in the segmented genome

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Generation of influenza A virus entirely from cloned c DNA Plasmids constructed for each of 8 segments of v RNA as cDNA in between RNA pol 1 promoter and terminator seq cDNA of each genes of viral proteins are cloned in diff eukaryotic expression plasmids Transfection of 293T human embrioyanic kidney cell line with different amounts of expression plasmids and RNA pol 1plasmids –total 17 diff plasmids used (Neumann,Watanabe etal,PNAS,1999)

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Plasmid construction All 8 seg of v RNA converted to cDNA cDNA amplified by PCR with primers containing BsmBI sites Clone in BsmBI sites of pHH21 pHH21 Has promoter and ter seq RNA pol1of mammals

Potential Applications of RG of viruses : 

Potential Applications of RG of viruses Structure –function study of individual viral genes…not by isolation in different system (reductionist approach) In viral infectious system .. their interaction with host cell To study molecular basis of pathogenicity of virus ex:- F protein cleavage by host protease determine the virulence of NDV(Ben &Olav J.viro,1999) RG provides direct evidence for correlation between HA cleavability and virulence of Avian Influenza A virus(Tisuke &Yoshihiro,J.Viro,1994)

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As live virus vaccines…. with deletion or alteration of virulent sequences Genetically engineered live attenuated Influenza A virus vaccine candidates(Neil&Peggy,J.vir,1997) Recombinant NDV expressing VP2 of IBD protects against both NDV and IBD(Zhuhui ,Subbiah and Abdul,J.Viro,2004) As viral vectors expressing foreign protein for vectored vaccines, gene therapy ,cancer treatment manufacture of biologicals Speculation of genetic vaccines using defective viral RNA genomes that replicate but not spread to neighboring cells

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Summary RG is a marked forward leap in understanding molecular biology and pathogenesis viruses and development of effective vaccines against many viral disease of animals and birds Development of effective vaccine for avian influenza to prevent the pandemics RG has expanded the horizons of recombinant DNA technology such as effective viral vaccine ,vectors ,biologicals and there are efforts to make further refinements in this new field

THANK YOU : 

THANK YOU

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