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Gene Targeting in Crop Plants A.K. CHHABRA

Gene Targeting:

Gene Targeting Gene targeting  is a  genetic  technique that uses homologous recombination to change an endogenous  gene . The method can be used to delete a  gene , remove exons, add a  gene , and introduce point mutations. Delete a gene Add a gene Delete an exon Introduce a point mutation Homologous recombination INTRODUCTION DEFINITION


Gene targeting allows to precisely integrate a transgene into a desired site in the genome, which ultimately has desired expression. Gene targeting can be used to selectively switch off, modify or replace genes. This technique has successfully been used in bacteria, yeasts and mice, but has yet to show its worth and full potential in plants Gene targeting requires the creation of a specific vector for each gene of interest. INTRODUCTION FEATURES


The process must be directed to the locus of choice. The process must be specific, i.e a desired sequence can be inserted or substituted at the target locus. The process should be efficient . Three criteria can be used as guideline INTRODUCTION CHARACTERSTICS OF PROCESS


Target“ refers to the gene or DNA segment subject to modification by the gene targeting method and the new gene designed to repair or replace it as the Donor. Target? Condition? Gene targeting requires homology between the Target and the Donor. Two types of gene targeting Site-specific recombination (SSR) and homologous recombination (HR). Types? INTRODUCTION


During meiosis, homologous recombination can produce new combinations of genes as shown here between similar but non identical copies of human chromosome 1. Homologous recombination is a type of genetic recombination in which nucleotide  sequences are exchanged between two . What is Homologous Recombination ? A Prerequisite for gene targeting INTRODUCTION

Site-specific recombination targeting (SSR):

Site-specific recombination targeting (SSR) Site-specific recombination targeting (SSR): It requires a site-specific recombinase, which can catalyze the recombination between target recognition sites, which are highly specific for the recombinase being used. Recombination and subsequent targeting depends upon both the homology and the orientation between target recognition sites. For example, depending upon the orientation of two Target recognition sites that flank a region of DNA, an inversion or an excision of the DNA can result. TYPES

RECOMBINASES Tyrosine recombinases (YR) and serine recombinases (SR), depending on their mechanism:

All enzymes recombine to target sites, which are either identical (subfamily A1) or distinct (phage-derived enzymes jn classes A2, B1 and B2). Whereas for A1 these sites have individual designations (FRT in case of Flp-recombinase, loxP for Cre-recombinase) the terms attP and attB (attachment sites on the phage and bacterial part, respectively) are valid in the other cases. In case of A1 we have to deal with short (usually 34 bp long) sites consisting of two (near-)identical 13 bp arms (arrows) flanking an 8 bp spacer (the crossover region, indicated by red line doublets). RECOMBINASES Tyrosine recombinases (YR) and serine recombinases (SR), depending on their mechanism TYPES RECOMBINASES


Tag and Exchange Strategy dependent on homologous recombination TYPES STRATEGY 1


Tag and Exchange Inversion, Excision and Integration strategies TYPES STRATEGY 2

Site-Specific Recombinase/Target recognition sites : Flp recombinase /FRT, Cre recombinase/lox, R recombinase /RS, and Gin recombinase/gix. :

Site-Specific Recombinase/Target recognition sites : Flp recombinase /FRT, Cre recombinase/lox , R recombinase /RS , and Gin recombinase/ gix . TYPES ALL RECOMBINASES

Flp-FRT recombination:

Flp -FRT recombination Flp- FRT  recombination  is a site-directed recombination technology used to manipulate an organism's DNA under controlled conditions  in vivo . It is analogous to Cre- lox  recombination but involves the recombination of sequences between short flippase recognition target ( FRT ) sites by the recombinase(Flp)derived from the 2µm plasmid of baker's yeast  Saccharomyces cerevisiae . TYPES Flp=FRT CONTD…………………………….

Flp-FRT recombination:

One of the major goals of site-specific recombination is to target desired genes to specific, well-characterized sites within the genome. We will distinguish between two types of targeting: Targeted displacement and targeted replacement. Targeted displacement occurs when the Donor gene displaces the Target gene, Targeted replacement occurs when the Donor gene replaces the target gene. Flp -FRT recombination TYPES Flp=FRT CONTD…………………………….

Gene construct:

Gene construct Apart from the gene of interest itself, a so-called promoter ("starter") and a terminator ("stop signal") are required for expression. In most cases, additional sequences are included, e.g. marker genes, which are also accompanied by a promoter and a terminator. The name "construct" is used because the sequences normally do not exist in this combination, but must be "put together" (constructed).   CONSTRUCT ANTIBIOTIC RESISTANCE GENE TRANSGENE (Gene of interest) TISSUE SPECIFIC PROMOTER SEQUENCE STOP SEQUENCE

Targeted displacement:

Targeted displacement ABCDEF ABCDEF

Recombination Mediated Cassette Exchange (RMCE).:

Recombination Mediated Cassette Exchange (RMCE). CASSETTE EXCHANGE RMCE

Targeted Replacement:

Targeted Replacement REPLACEMENT TR

Homologous recombination targeting (HR):

Homologous recombination targeting (HR) Originally demonstrated by Oliver Smithies (Smithies et al . 1985) Occurs when sequences within the genome are recombined with homologous introduced sequences. HR targeting relies on the combination of homology between Target and Donor and the endogenous recombination machinery of the plant or animal. RECOMBINATION HR CONTD……………………………


When one wants to replace one allele with an engineered construct but not affect any other locus in the genome, then the method of choice is homologous recombination. To perform homolgous recombination, one must know the DNA sequence of the gene you want to replace. With this information, it is possible to replace any gene with a DNA construct of your choice. Homologous recombination targeting (HR) RECOMBINATION HR CONTD……………………………


Diagram of gene targeted for replacement by an engineered construct. The coding sequence is illustrated by the box with flanking upstream and downstream DNA sequences provided. The arrows pointing away from the targeted gene represent the continuous chromosomal DNA. Diagram of engineered construct that will be used to replace the wild-type allele. The upstream and downstream flanking DNA sequences are identical to those which flank targeted locus. The negative marker tk is shown in to the right of the region of sequence similarity. ABCDEF ??????? ?????????????? HR


Diagram of alignment of DNA just prior to homolgous recombination. Amazingly, the DNA construct finds its way into the nucleus and then aligns itself with the targeted locus. Diagram of the final products of homologous recombination. The chromosome now contains a portion of the flanking DNA as well as all of the engineered construct which has taken the place of the original allele. The original allele has been recombined into the construct and thus is lost over time.. ABCDEF


If the targeting vector aligns in a non-homologous region of the genome, then recombination is random and the negative selection marker may become incorporated into the genome. End products of non-homologous recombination. The positive and negative selection markers are incorporated into the chromosome. so gancyclovir will kill cells with modified chromosomes as shown.

Methods of gene targeting:

Methods of gene targeting Vector-mediated or indirect gene transfer. Vectorless or direct gene transfer Chemical mediated gene transfer Microinjection Electroporation Particle gun/Particle bombardment Conjuction Liposome mediated gene transfer or Lipofection –stem cell ABCDEF METHODS

Vector-mediated or indirect gene transfer :

Vector-mediated or indirect gene transfer Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used . Natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues The foreign gene is cloned in the T-DNA region of the Ti plasmid in place of unwanted sequences. . METHODS VECTOR-MEDIATED


Vector-mediated or indirect gene transfer METHODS VECTOR-MEDIATED

Vectorless or direct gene transfer :

Vectorless or direct gene transfer In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are: Chemical mediated gene transfer Chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts. Calcium phosphate is also used to transfer DNA into cultured cells. METHODS VECTORLESS


Microinjection : where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes , eggs, and the cells of early embryo. Electroporation : Involves a pulse of high voltage applied to protoplasts/ cells/ tissues to make temporary pores in the plasma membrane which facilitates the uptake of foreign DNA. The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus. METHODS MICROINJECTION AND ELECTROPOATION


Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/micro projectile gun). Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adhere to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells. METHODS


METHODS PARTICLE GUN Helios Gene Gun Standard Gene Gun Mechanism

Applications of gene targeting:

Applications of gene targeting Butefecanil herbicide resistance by Gene targeting in Arabidopsis. To correct mutated genes, with obvious application in gene therapy. To introduce mutations in gene. Knockout mouse APPLICATIONS

GT in Arabidopsis:

GT in Arabidopsis Endogenous gene PPO (protoporphyrinogenoxidase) catalyses oxidation of protoporphyrinogen IX to protoporphyrin, the last common step in heme and chlorophyll biosynthesis. The PPO protein residing in chloroplasts (cPPO) is the target of several herbicides (e.g. Butafenacil), which competitively block the enzyme. After inhibition of cPPO, accumulating protoporphyrinogen IX leaks to the cytoplasm where it is rapidly oxidized to protoporphyrin by non-specific peroxidases. ARABIDOPSIS

GT in Arabidopsis:

In the light, this generates reactive singlet oxygen, which results in rapid cell damage. Two simultaneous mutations introduced into Arabidopsis PPO cDNA expressed as a transgene render plants highly resistant to PPO-inhibiting herbicides. One mutation is a single bp change at codon 305 (TCA to TTA) leading to an amino acid change, S to L. The second one is at codon 426 which leads to one amino acid change and generates a novel restriction site (CTAC to CTAG). GT in Arabidopsis ARABIDOPSIS


GT in Arabidopsis ARABIDOPSIS

Knockout Mouse :

Knockout Mouse A knockout mouse has had both alleles of a particular gene replaced with an inactive allele. This is usually accomplished by using homologous recombination to replace one allele followed by two or more generations of selective breeding until breeding pair are isolated that have both alleles of the targeted gene inactivated or knocked out. Knock out mice allow investigators determine the role of a particular gene by observing the phenotype of individuals that lack the gene completely. APPLICATION KNOCKOUT MOUSE


STEPS Step 1. Isolate developing embryo at blastocyst stage. This embryo is from a strain of mice with grey fur. Step 2. Remove embryonic stem cells from grey-fur blastocyst. Grow stem cells in tissue culture. Step 3. Transfect stem cells with homologous recombination construct. Select for homologous recombination by growing stem cells in neomycin and gancyclvir


Step 4. Remove homologously recombined stem cells from petri dish and inject into a new blastocyst that would have only white fur. Step 5. Implant several chimeric blastocysts into pseudo-pregnant, white fur mouse. Step 6. Mother will give birth to a range of mice. Some will be normal white fur mice but others will be chimeric mice. Chimeric mice have many of their cells from the original white fur blastocyst but some of their cells will be derived from recombinant stem cells. Fur cells from recombinant stem cells produce gray patches which are easily detected. STEPS


Step 7. Mate the chimeric mice with wild-type white fur mice. If the gonads of the chimeric mice were derived from recombinant stem cells, all the offspring will have gray fur. Every cell in gray mice are heterozygous for the homologous recombination. STEPS Step 8. Mate heterozygous gray mice (+/ H) and genotpye the gray offspring. Identify homozygous recombinants (H / H) and breed them to produce a strain of mice with both alleles knocked out. The pure breeding mouse strain is a "knockout mouse".

Enzymes for increasing GT efficiency:

Enzymes for increasing GT efficiency To enhance the efficiency of gene targeting, a chromosome break is created at the site of modification (the target). An enzyme called a zinc finger nuclease (ZFN) is used to generate the chromosome break. Zinc finger arrays can be designed to recognize any site in the plant genome, thereby making it possible modify any chromosomal sequence. Another approach, is to create a break in the DNA strand (double-strand break) at a precisely defined site in the plant genome using a rare cutting enzyme called "I-SceI". This increases the likelihood of homologous recombination occurring at this site. EFFICIENCY



Comparison with gene trapping :

Comparison with gene trapping Gene trapping is based on random insertion of a cassette while gene targeting targets a specific gene. Cassettes can be used for many different things while the flanking homology regions of gene targeting cassettes need to be adapted for each gene. Particular gene of interest may not be mutated. Cannot be used for genes which are permanently switched off. COMPARISON


Limitations Gene targeting experiment is laborious and time-consuming. Demand elaborate techniques for the manipulation of DNA, cells and embryos, and it often takes more than one year from the design of constructs to generation of animals. One of the limiting steps is the generation of ‘gene targeting vectors’, since the design of a targeting vector must fulfill several requirements Gene targeting is inefficient in most higher eukaryotes, including plants, because the frequency of random DNA integration exceeds that of homologous integration by three to four orders of magnitude. LIMITATION



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