Gene Transferee & Applications of Genetics PDF

GENE TRANSFEREE & APPLICATIONS OF GENETICS Dr Ahmed Noby Amer  GENE TRANSFEREE ▪ Horizontal gene transfer, is a process in which an organism transfers genetic material to another organism that is not its offspring. ▪ There are three mechanisms of horizontal gene transfer in bacteria: ▪...

GENE TRANSFEREE & APPLICATIONS OF GENETICS Dr Ahmed Noby Amer  GENE TRANSFEREE ▪ Horizontal gene transfer, is a process in which an organism transfers genetic material to another organism that is not its offspring. ▪ There are three mechanisms of horizontal gene transfer in bacteria: ▪ transformation ▪ Transduction ▪ conjugation.  1. CONJUGATION ▪ Genetic recombination in which there is a transfer of DNA from a living donor bacterium to a living recipient bacterium by cell-to-cell contact. it typically involves a conjugation or sex pilus. ▪ Conjugation is encoded by plasmids or transposons. It involves a donor bacterium that contains a conjugative plasmid and a recipient cell that does not. A conjugative plasmid ▪ is self-transmissible, in that it possesses all the necessary genes for that plasmid to transmit itself to another bacterium by conjugation. ▪ Many conjugative plasmids and conjugative transposons possess can transfer DNA not only to like species.  1. CONJUGATION ▪ 1. conjugation involves a conjugation pilus (sex pilus or F pilus) on the donor bacterium binding to a recipient bacterium lacking a conjugation pilus. ▪ 2. A series of membrane proteins coded for by the conjugative plasmid then forms a bridge and an opening between the two bacteria, now called a mating pair. ▪ 3. Nuclease breaks one strand of the plasmid DNA the plasmid and that nicked strand enters the recipient bacterium. The other strand remains in the donor cell. Both the donor and the recipient plasmid strands then make a complementary copy of themselves.  2. TRANSDUCTION ▪ Transduction involves the transfer of a DNA fragment from one bacterium to another by a bacteriophage. ▪ There are two forms of transduction: ▪ A. generalized transduction ▪ B. specialized transduction.  2. TRANSDUCTION ▪ Generalized transduction ▪ Occur during the replication of lytic and temperate bacteriophages, occasionally the phage capsid accidently assembles around a small fragment of bacterial DNA. ▪ When this bacteriophage, called a transducing particle, infects another bacterium, it injects the fragment of donor bacterial DNA it is carrying into the recipient.  2. TRANSDUCTION ▪ Specialized transduction ▪ occur occasionally during the lysogenic life cycle of. During spontaneous induction temperate bacteriophage, a small piece of bacterial DNA may sometimes be exchanged for a piece of the bacteriophage genome, which remains in the bacterial nucleoid. This piece of bacterial DNA replicates as a part of the bacteriophage genome and is put into each phage capsid. The bacteriophages are released, adsorb to recipient bacteria, and inject the donor bacterium DNA/phage DNA complex into the recipient bacterium where it inserts into the bacterial chromosome.  3. TRANSFORMATION ▪ Transformation is a form of genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and is exchanged for a piece of DNA of the recipient. The recipient for the DNA are called Competent cells. ▪ A few bacteria, such as Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneomophila, Streptococcus pneumoniae, and Helicobacter pylori tend to be naturally competent and transformable, In some other bacteria transformation is induced artificially, this helps in introduction of an accessory piece of DNA (Plasmid) to such bacteria Ex E.coli  3. TRANSFORMATION ▪ Competent cells preparation: ▪ A. E.coli cells treated with cold solutions containing Ca+ ions were rendered susceptible to take up exogenous DNA.Ca Cl2 has four major factors: ▪ 1. It has a divalent cation, helps to grab (attract and hold )two phospholipids making them twice as big and more rigid slowing down the membrane motion, ▪ 2. Has a cold solution that further helps to decrease the membrane fluidity. ▪ 3. It is also helping the DNA plasmid itself to shrink down, ▪ 4. It is hypertonic to the media causing the bacteria t loses some of the water that it has in there and shrink.  3. TRANSFORMATION ▪ Competent cells preparation: ▪ B. The mixture is then heat shocked at 42°C for 90 secs to induce enzymes involved in repair of DNA and other cell constituents ▪ Small change in temperatures causes the bacteria to swell very rapidly. As it swells, it is very likely to crack or to have small fissures open on the surface. This usually occurs on a part of the bacteria known as the adhesion zone. ▪ These little cracks occur and it is going to pull in water very quickly through those cracks. As it does so, it is very likely to pull in a plasmid. ▪ As a result of this heat shock, it has to swell up, crack, suck in a plasmid, and then stop itself from rupturing completely, and reseal.  HOW DO MINUTE MICROORGANISMS ACTUALLY RESIST ANTIMICROBIAL ACTIONS? WHAT ENABLES THEM TO DO THIS? ▪ The influence exerted by some factor (such as an antimicrobials) on natural selection to promote one group of organisms over another. In the case of antimicrobial resistance, antimicrobial cause a selective pressure by killing susceptible bacteria, allowing antimicrobial-resistant organism to survive and multiply.  HOW DO MINUTE MICROORGANISMS ACTUALLY RESIST ANTIMICROBIAL ACTIONS? WHAT ENABLES THEM TO DO THIS? ▪ The acquisition of resistant phenotype takes place by ▪ 1. Mutation ▪ 2. Resistance Gene transfer  HOW DO MINUTE MICROORGANISMS ACTUALLY RESIST ANTIMICROBIAL ACTIONS? WHAT ENABLES THEM TO DO THIS? ▪ 1. Mutation: Any change in a single base pair may lead to a corresponding change in one or more of the amino acids for which it codes, which can then change the enzyme or cell structure that consequently changes the affinity or effective activity of the targeted antimicrobials. 2. Horizontal gene transfer: Many of the antibiotic resistance genes are carried on plasmids, transposons or integrons that can act as vectors that transfer these genes to other members of the same bacterial species, as well as to bacteria in another genus or species. Detection of Genotypic resistance is carried out by sequencing analysis for Mutation & by PCR for Gene transfer  DETECTION OF RESISTANT GENES ▪ PCR Polymerase chain reaction (PCR) ▪ Polymerase chain reaction is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, a single copy of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. ▪ The technique is used for multiple application including diagnosis of microbial infection (viral)  DETECTION OF RESISTANT GENES ▪ PCR is carried out in three steps ▪ 1. Denaturation: at 95 oC : increasing the temperature to separate the DNA double strands ▪ 2. Annealing ( 50 oC to 60 oC) decreasing temperature allowing the primer to bind to DNA strands ▪ 3. Extension: at 72 oC: when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.  DETECTION OF RESISTANT GENES ▪ *Primers are single strands of DNA sequence that are around 20 to 30 bases in length. Serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA ▪ Taq polymerase ▪ thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus ▪ Tutorial 5 ▪ https://goo.gl/forms/q 42wzsXx5iQKzSBg1  GENE THERAPY ▪ A branch of Regenerative Medicine, an emerging field that involves the "process of replacing, engineering or regenerating human cells, tissues or organs to restore or establish normal function” ▪ Gene therapy is the delivery of therapeutic gene into a patient's cells to treat disease. ▪ Cell therapy is the delivery of intact, living cells into a patient to treat disease.  GENE THERAPY ▪ There are several approaches for correcting faulty genes; the most common being the insertion of a normal gene into a specific location within the genome to replace a nonfunctional gene. Gene therapy is classified into the following two types: ▪ 1. Somatic Gene Therapy ▪ 2. Germ Line Gene Therapy  TYPES OF GENE THERAPY ▪ 1. Somatic Gene Therapy ▪ In somatic gene therapy, the somatic cells of a patient are targeted for foreign gene transfer. In this case the effects caused by the foreign gene is restricted to the individual patient only, and not inherited by the patient's offspring or later generations. ▪ 2. Germ Line Gene Therapy ▪ Here, the functional genes, which are to be integrated into the genomes, are inserted in the germ cells, i.e., sperm or eggs. Targeting of germ cells makes the therapy heritable.  GENE THERAPY STRATEGIES ▪ A. Gene Augmentation Therapy (GAT) ▪ simple addition of functional alleles is used to treat inherited disorders caused by genetic deficiency of a gene product, e.g. GAT has been applied to autosomal recessive disorders  GENE THERAPY STRATEGIES ▪ B. Targeted Killing of Specific Cells ▪ It involves utilizing genes encoding toxic compounds (suicide genes), or prodrugs (reagents which confer sensitivity to subsequent treatment with a drug) to kill the transfected/ transformed cells. This general approach is popular in cancer gene therapies.  GENE THERAPY STRATEGIES ▪ C. Targeted Inhibition of Gene Expression ▪ To block the expression of any diseased gene or a new gene expressing a protein which is harmful for a cell. This is particularly suitable for treating infectious diseases and some cancers.  GENE THERAPY STRATEGIES ▪ D. Targeted Gene Mutation Correction ▪ The ability to change an organism's DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. These strategies are possible for gene therapy.( CRISPR)  GENE THERAPY STRATEGIES ▪ D. Targeted Gene Mutation Correction  METHODS OF GENE TRANSFER ▪ There are mainly two approaches for the transfer of genes in gene therapy: ▪ 1. In vitro gene transfer Transfer of genes into patient cells outside the body (ex vivo gene therapy) ▪ 2. In vivo gene transfer Transfer of genes directly to cells inside the body (in vivo).  METHODS OF GENE TRANSFER ▪ A. In vitro Gene transfer protocol ▪ 1. Remove host cells ▪ 2. Expand by cell culture ▪ 3. Transfect with new gene ▪ 4. Establish stable transfection ▪ 5. Return to host  METHODS OF GENE TRANSFER ▪ A. In vitro Gene transfer protocol ▪ Main indications: ▪ Defects affecting blood cells, e.g. severe combined immune deficiency due to adenosine deaminase malfunction. ▪ Some possibilities using fibroblasts, hepatocytes and myoblasts, but involve greater difficulties than with blood cells but: ▪ Difficult to get prolonged gene expression and ▪ establish a genetically altered cell population ▪ Number of suitable target diseases is limited.  METHODS OF GENE TRANSFER ▪ A. In vivo Gene transfer protocol ▪ Use of any of the normal parenteral delivery routes for gene therapy using, viral or non- viral vectors. ▪ Has all the problems of in vitro delivery, and in addition has to fulfil following criteria: ▪ Avoid uptake of RES ▪ Avoid immunogenic responses if more than one administration is required. ▪ Target to relevant organ or tissue  METHODS OF GENE TRANSFER ▪ A. In vivo Gene transfer protocol ▪ Viral Vectors ▪ Where some of the DNA involved in viral replication is removed and replaced by a gene cassette. ▪ Viral vectors are very efficient at transfecting cells – have evolved machinery to deliver DNA effectively. However there are worries about: ▪ Disabled viruses regaining virulence, ▪ Viruses are very immunogenic ▪ Viral therapy has resulted directly in a death and two cases of leukemia.  METHODS OF GENE TRANSFER ▪ A. In vivo Gene transfer protocol ▪ Non-Viral Vectors ▪ Non-Viral vectors easier to manufacture, scale-up and quality control ▪ Becomes like any other drug delivery problem - familiar ground for the pharmaceutical industry.