Gene Silencing

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GENE SILENCING

GENE SILENCING:

GENE SILENCING Transgenes when inserted into plants can either promote the expression or suppression of homologous genes. The suppression of homologous genes by the transgenes is called GENE SILENCING.

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It is a form of epigenetic modification. The silent gene can undergo spontaneously as well as developmentally regulated reactivation, called as RESETTING . It is of two types : - transcriptional gene silencing. - post transcriptional gene silencing. Transcriptional silencing is due to promoter methylation, while post transcriptional silencing can involve methylation of coding sequence.

TYPES OF GENE SILENCING:

TYPES OF GENE SILENCING HOMOLOGY DEPENDENT GENE SILENCING – It occurs when no: of copies of transgenes are integrated at same locus or in different positions within the genome. Crossing with hypermethylated homologous sequences occurs. E.g: NOS synthase gene silencing in tobacco

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POSITION EFFECTS Gene integrated in hypermethylated inactive region become silenced. ENVIRONMENTAL STRESS It may be a natural defense mechanism of the plant against the foreign DNA followed by methylated and thus get silenced. It can be duplicated in tissue culture by addition of propionic acid or butyric acid.

ANTI SENSE GENE SILENCING:

ANTI SENSE GENE SILENCING It involves inversion of coding region b/w promoter and terminator sequences. Both sense and anti sense strand get transcribed. RNA so synthesised binds to RNA transcribed from normal gene. RNA – RNA complex can’t be translated into functional protein, thus gene expression is switched off.

POST TRANSCRIPTIONAL GENE SILENCING:

POST TRANSCRIPTIONAL GENE SILENCING In this promoters are active , and the genes transcribed but mRNA fails to accumulate. It is the ability of exogenous dsRNA to suppress the expression of gene having homologous sequence as the dsRNA.

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dsRNA produced by - transcription of inverted repeats - viral replication - transcription of RNA by RNA dependent RNA polymerase. This dsRNA promotes cleavage of homologous mRNA.

CO-SUPPRESSION:

CO-SUPPRESSION Introduction of transgene homologous to endogenous gene resulted in plants with both genes suppressed. It resulted in degradation of both endogenous and transgene mRNA.

SMALL INTERFERING RNA:

SMALL INTERFERING RNA It is involved in PTGS. These are 21-25 nt fragments which bind to complementary portion of target mRNA and tag it for degradation. A single bp difference b/w the target mRNA and siRNA is enough to block the process.

STEPS TO MINIMISE TRANSGENE SILENCING:

STEPS TO MINIMISE TRANSGENE SILENCING Transformation vector should not have duplicated sequences. Each gene construct of vector should have different promoter and polyadenylation signal. Transgene should not contain sequences that interfere with mRNA. Transgene should be integrated in a single copy , and away from chromosomal locations having hypermethylation.

GENE SYNTHESIS:

GENE SYNTHESIS Two methods Chemical synthesis RNA directed DNA polymerase synthesis

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If the detailed structure of gene is known the gene can be synthesized by chemical method ( KHORANA ) Chemical assembly of nucleotides into short stranded DNA fragments called as oligonucleotides. Enzymatic assembly of oligonucleotides into ds clonable DNA.

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If the detailed structure is not known RNA directed DNA polymerase enzyme is utilized for invitro synthesis of gene.cDNA is synthesized from the mRNA. mRNA of desired protein is used as template to synthesize cDNA using enzyme reverse transcriptase.

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Total RNA is extracted ↓ Poly A sequence is added to 3’ end of desired mRNA ↓ cDNA is formed having hair pin loop at the end ↓ Hair pin loop is trimmed using enzyme S 1 nuclease

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GENE CLONING

GENE CLONING:

GENE CLONING It is also called as recombinant DNA technology or molecular cloning. It is for transfer of genetic materials from one organism to other. It is for getting more copies of a particular gene in desired host cell.

STEPS IN GENE CLONING:

STEPS IN GENE CLONING Selection of suitable vector generating DNA fragments Joining foreign DNA to the appropriate vector to form recombinant DNA Introduction of recombinant DNA into host cell Selection of transformed cells Analysis of clones Expression of inserted gene into target cell

VECTORS:

VECTORS These are carrier DNAs into which foreign DNA or gene of interest are spliced to make recombinant DNA VEHICLE DNAs. It is generally of two types- 1) Cloning vectors –used for obtaining million copies of cloned DNA segment. The cloned genes in these vectors are not expected to express at transcription or translation levels.

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2) Expression vectors- it allows expression of cloned gene, to give the product. Important parts of a vector are origin of replication and promoter region. It should have several markers like antibiotic resistance gene. Unique restriction site for cloning , located in a position where the inserted DNA can expressed efficiently.

PLASMIDS:

PLASMIDS These are extra chromosomal self replicating double stranded , closed circular DNA molecules present in the bacterial cells.

Plasmid contd.:

Plasmid contd. Can be easily isolated from the cells. Available in multiple copies Can be reintroduced into the bacterial cells.

BACTERIOPHAGES:

BACTERIOPHAGES These are viruses that infect bacteria. It is having linear DNA , so a single break will create two fragments. Foreign DNA can be inserted in b/w the fragments.

λ PHAGE:

λ PHAGE contain double stranded linear DNA. Advantages -large sized DNA can be cloned. -recombinant DNA can be easily multiplied.

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COSMID They are hybrid vectors derived from plasmids. It contains cos site of λ phage, which is needed for binding and cleavage. PHASMID They are linear molecules prepared artificially to combine desirable features of both plasmid and phage.

SHUTTLE VECTORS:

SHUTTLE VECTORS They are capable of replication in cells of two different species. It contains two origin of replication. It helps in transfer of genes b/w unrelated species

GENERATING DNA FRAGMENTS :

GENERATING DNA FRAGMENTS Restriction endonucleases are used for cleaving the DNA molecule. It can be of 3 types Type 1 Restriction endonucleases –cleave only one strand of DNA Type 2 Restriction endonucleases –cleave both the polynucleotide chains within or near the palindromic sequence Type 3 Restriction endonucleases –cleave double stranded DNA at well defined sites.

CLEAVAGE STYLES:

CLEAVAGE STYLES 1) sticky end style – target sequence is asymmetrical, staggered cuts in two strands of DNA are some nucleotides apart producing complementary SS protruding ends. 2) Blunt end style – In this cut is made across both strands of DNA at the same position.

INSERTION OF TARGET DNA INTO VECTORS:

INSERTION OF TARGET DNA INTO VECTORS Sticky end ligation : complementary sticky ends of foreign DNA and vector join by complementary base pairing by renaturation and annealing. Blunt end ligation : blunt ends of both foreign DNA fragment and its cloning vectors join each other with the help of DNA ligases. Homopolymer tailing method : sequence of same nucleotide is added to 3’ end . Terminal transferase enzyme is involved.

INTRODUCTION OF RECOMBINANT DNA INTO HOST CELL:

INTRODUCTION OF RECOMBINANT DNA INTO HOST CELL TRANSFORMATION TRANSDUCTION ELECTROPORATION LIPOSOMES MICROINJECTION MICROPROJECTILE

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TRANSFORMATION is the uptake of foreign DNA by a cell from its surroundings. TRANSFECTION is the introduction of foreign DNA into bacteria by bacteriophage ELECTROPORATION is the introduction of foreign DNA into cell by exposure to electric pulse.

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LIPOSOME are small lipid bags in which large plasmids are enclosed, these liposomes are induced to fuse with protoplasts and thus used for gene transfer. MICROINJECTION is the delivering of DNA with the help of micro manipulators. MICROPROJECTILE is used for transferring DNA coated with tungsten or gold into plant cell at very high speed.

SCREENING AND SELECTION OF TRANSFORMED CELLS:

SCREENING AND SELECTION OF TRANSFORMED CELLS It is achieved with the help of selectable marker genes or reporter genes present in the recombinant vectors. Marker – antibiotic resistance Reporter gene – beta galactosidase

ANALYSIS OF CLONE:

ANALYSIS OF CLONE - Clone containing the insert is analysed with same restriction enzyme and sample is subjected to electrophoresis. EXPRESSION OF FOREIGN DNA INSIDE CLONE - Strong promoter is needed for expression of foreign DNA

APPLICATIONS:

APPLICATIONS Cure of diseases Production of vaccines and drugs Biosynthesis of interferones Industrial and agricultural applications Bioremediation

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