It s All in the Hands Genetic Engineering

Transcription

1 It s All in the Hands Genetic Engineering Genetic Engineering Genetic Engineering is the technique of modifying the genome of an organism by using recombinant DNA technology. Recombinant DNA (rdna) technology isolates a specific DNA segment (gene/genes) from one organism to insert it in another DNA molecule at a desired position. Scientists can identify and remove a desirable gene virtually from any living organism and insert it into any other organism. This rdna technology is important for commercial and pharmaceutical products. Genetic engineering includes diverse techniques: isolation, purification, and quantitation cellular DNA restriction endonuclease digestion construction of recombinant (rdna) molecules; cloning vectors; introduction of vectors into host cells to produce clones; construction of gene libraries; selection of specific clones; screening gene libraries for gene of interest; polymerase chain reaction (PCR) to amplify specific DNA sequences or genes in vitro; site-directed mutagenesis; transgenic technology. Recombinant DNA (rdna) Technology Recombinant DNA (rdna) is constructed in a test tube by covalently joining (recombining) DNA molecules from different origins. The DNA molecule from either a bacterial or viral source is called the vector as it can enter an appropriate cell to transfer the desired gene. The objective is to amplify the transferred gene through the multiplication of the transformed bacterium and then to get the desired product of the transferred gene in large amount. The gene to be inserted in an rdna needs to be isolated from a full length DNA molecule and again a nick is to be made in vector DNA molecule for insertion of the gene. Restriction endonucleases (enzymes) act as molecular scissors to provide cuts in intact DNA molecules. The enzymes cut at sites with specific nucleotide sequences Bacteria normally use these as defense against DNA from bacteriophage. These enzymes recognize specific nucleotide sequences in the DNA and cleave the

2 phosphodiester bond in the sugar phosphate backbone between nucleotides This is done within or close to recognition site which are mostly 4-base and 6-base long palindromes that is, nucleotide-pair sequences read the same forward There are many restriction enzymes that cut DNA at specific base sequences the recognition sequence, or restriction site. Restriction enzymes do not cut bacterial DNA because the recognition sequences are modified. Methylases add methyl groups after replication; makes sequence unrecognizable by restriction enzyme. Bacterial restriction enzymes can be isolated from cells. DNA from any organism will be cut wherever the recognition site occurs. EcoRI (from E. coli) cuts DNA at a particular sequence. After DNA is cut, fragments of different sizes can be separated by gel electrophoresis. Mixture of fragments is place on a well in a porous gel. An electric field is applied across the gel. Negatively charged DNA fragments move towards positive end. Smaller fragments move faster than larger ones. Electrophoresis provides information on: Size of fragments. Fragments of known size provide comparison. Presence of specific sequences. These can be determined using probes. Cloning Vectors for Amplification of Recombinant DNA Molecules A cloning vector needs to have three essential features or components: An origin of replication, which allows replication of inserted DNA along with the replication of the vector. A selectable marker gene--to indicate whether the rdna entered in the host cell. This is usually a gene that confers antibiotic resistance to the host cells A unique restriction endonuclease cleavage site (insertion site) known as the multiple cloning site (MCS). This is found far away from origin of replication site and selectable marker gene. This is the site for introducing a foreign DNA piece. Different types of Vectors Plasmid Vectors Bacteriophage Cosmid Vectors- Contains cos sequence from lambda phage. This allows enzymes produced by lambda to package the cosmid into the capsid. This allows for larger pieces of DNA to be inserted. Phagemid vectors- Similar to cosmids except that: cos sites are absent and origin of replication of F1 phage is present. Absence of antibiotic resistance genes In order for transduction to occur, sex pili are required. Eukaryotic and Shuttle Vectors (can replicate in both E. coli and eukaryotic species)

3 Artificial chromosomes- yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) Plasmids Plasmids have all these characteristics: Plasmids are small, many have only one restriction site. Genes for antibiotic resistance can be used as reporter genes. They have an origin of replication and can replicate independently. Plasmids can be used for genes of 10,000 bp or less. Most eukaryote genes are larger than this. Viruses can be used as vectors e.g., bacteriophage. The genes that cause host cell to lyse can be cut out and replaced with other DNA. Bacterial plasmids don t work for yeasts because the origins of replication use different sequences. A yeast artificial chromosome (YAC) has been created: contains yeast origin of replication, plus yeast centromere and telomere sequences. Also contains artificial restriction sites and reporter genes Construction of Genomic DNA Libraries Entire genome of an organism is cleaved with a restriction endonuclease into many fragments which are then accommodated into many appropriate cloning vectors. The collection of such recombinant DNA clones constitute genomic or DNA library of an organism. Isolation and Purification of RNA For the purpose of genetic engineering, RNA isolation is required for purification of mrna which is used for the preparation of complementary DNA (cdna) for making a gene library. Similar to DNA isolation strategies, RNA isolation involves same basic steps cell lysis, removal of contaminating biomolecules, and precipitation of RNA. Complete inhibition of RNase activity is must for mrna extraction. Construction of Complementary DNA Libraries cdna Libraries The clones having all cdna inserts from an organism constitute the cdna library for the organism. The total number of cdna clones of an organism is supposed to be smaller than the number of clones for genomic DNA library of the same organism Introduction of rdna-vectors into Bacterial Cells Chemical: CaCl2 and Heat Shock Technique Physical: Transformation by Electroporation Phage: Mediated Transfer of rdna

4 Selection of Transformed Cells Harboring rdna It is important in rdna technology to select the host cells that have taken up the DNA construct (i.e., transformed cells). Selectable marker genes (antibiotic drug resistant genes on plasmids, lac z gene marker, and others) help to select easily positive recombinant clone(s). Screening DNA Libraries (Clones) for Genes of Interest Essentially, a DNA library for an organism has a huge number of clones depending on size of the genome of the organism. Now the question is how to identify a clone with a single DNA sequence of interest out of a million or more clones. Hybridization Polymerase Chain Reaction Hybridization Each transformed host cell of a library will contain only one vector with one insert of DNA. A filter is placed on top of colonies growing on an agar plate, transferring (blotting) the colonies onto the membrane filter. The cells are then lysed, the DNA denatured and then labelled probes are added. The labelled probes will bind to complementary regions of DNA. Unhybridized probes are washed off and autoradiography is used to visualize the hybridized DNA Polymerase Chain Reaction (PCR) To make multiple copies of a piece of DNA enzymatically Used to Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens Screen DNA libraries Taq polymerase Thermus aquaticus Can withstand 95 C Pfu polymerase Pyrococcus furiosus Higher fidelity Site-Directed Mutagenesis Induction of a mutation at a nucleotide (point mutation) or at several sites of a gene to alter the function of a gene including its protein product is now possible. Such defined alteration at a site in a gene sequence of a clone in vitro is known as site-directed mutagenesis (or site-specific mutagenesis).

5 Transgenic Organisms (Plants and Animals) When one or more, synthetic, modified, and foreign genes are introduced into eukaryotes plants and animals, the resulting hosts are called transgenic organisms. Usually a transgenic plant or animal is a fertile organism that carries an introduced gene(s) in its germ line so that the new gene(s) becomes part of the genome of the next generations. Examples of Transgenic Plants and their Utility Bacillus thuringiensis Gram-positive soil bacterium cry gene which encodes a protein that is toxic to many kinds of insects. This endotoxin is named Bt toxin Transfer of cry gene to different plant species gives them possession of an endogenous insecticidal property. Bt toxin disrupts gut epithelium of the insects leading to their death. The toxin is active against lepidopteran insects, that normally damage corn and cotton (Bollworm larva), dipteran, and coleopteran insects. Examples of Transgenic Plants and their Utility Herbicide resistance in agronomic crops A gene that encodes the enzyme nitrilase, from the soil bacterium Klebsiella ozaenae. inactivates bromoxynil (herbicide) by removing nitrogen before the herbicide can act. The removed nitrogen may be utilized by the plant for its own growth. The transgenic tobacco plants expressing the nitrilase gene are resistant to the toxicity of bromoxynil. Thus, weeds in a field of the transgenic tobacco plantation can be killed by spraying bromoxynil without affecting the tobacco plants. Forced Introduction of DNA Sequences with the Help of Gene Gun Bombardment of microprojectiles coated with DNA has been developed to introduce transgene into plant cell suspensions, callus cultures, meristematic tissues, immature embryos, coleoptiles, and pollens of a wide range of plants including dicots, monocots, and conifers. This method also enables to deliver genes into organelles like chloroplasts and mitochondria. Embryonic Stem Cell Technology to Raise Transgenic Animals Embryonic stem cells (ES cells) are a group of inner cell mass (ICM) found in the blastula stage of mouse or mammalian embryos. The cells are pluripotent; each of them can differentiate into many kinds of tissue. The ES cells can be cultured in vitro, transfected or injected with DNA, and then introduced into other developing blastocysts. When ES cells contribute to the formation of chimeric germ line tissue, the introduced foreign DNA gets a chance of being transmitted to the next generation. Breeding of such a chimeric mouse is likely to establish a transgenic strain.

6 Somatic Cell Nuclear Transfer (SCNT) in an Enucleated Ovum Dolly was produced by transfer of the nucleus from an epithelial cell of udder (mammary gland) of a Finn Dorset ewe (white faced) in an enucleated egg from a Blackface ewe This manipulated egg was stimulated to divide in vitro and then the blastocyst was implanted in the uterus of another Blackface ewe for gestation and birth. Dolly was white faced like its genetic (nucleus contributing) mother.