Lecture 17 - Genetic Engineering Genetic engineering is the...

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Unformatted text preview: Genetic Engineering Genetic engineering is the artificial alteration of genetic material which describes activities that include manipulation of DNA, introduction of changes into specific somatic cells or the germline of an organism. Genetic Engineering Elasri USM BSC476/576 Elasri USM BSC476/576 Cloning To study a DNA fragment (e.g., a gene), it needs to be: Stored Amplified Purified Cloning of DNA into a vector allows us to store, amplify and purify a gene. A cloning vector is a plasmid or phage that is used to 'carry' inserted foreign DNA for the purposes of producing more material or a protein product. The vector usually carries the origin of replication that will let it perpetuate itself in an appropriate host, most often E. coli or S. cerevisiae. cerevisiae. Elasri USM BSC476/576 Cloning New DNA can be introduced into the genome of an organism. The DNA of interest is cloned and delivered to the target cell. The cloned DNA is then integrated into the host chromosome by a recombination event. Elasri USM BSC476/576 Delivery of DNA There are several ways to deliver DNA into a target cell: Electroporation: electric filed used to shock cells which makes them take up DNA in the solution. Viral infection: use a viral vector that uses the viral infective process to enter the cell. Liposomes: small spheres made from artificial Liposomes: membranes that carry DNA. Liposomes fuse with the plasma membrane and release their content. Microinjection: very fine needle to puncture the cell membrane nd inject DNA into the cytoplasm, or directly into the nucleus. Gene gun: shoots DNA-coated nanospheres into DNAcells. Used in cells with thick cell walls (i.e. yeast, plants). Elasri USM BSC476/576 Cloning Vectors Vectors are used for different purposes and can be separated into several categories into depending on their properties. Plasmids can be easily engineered to carry long sequences of donor DNA in donor bacteria. Bacteriophages can be used in bacteria but have the disadvantage that only a limited amount of DNA can be packaged into the viral coat. Cosmid which propagates like a plasmid but uses the packaging mechanism of mechanism phage lambda to isolate the DNA (max capacity 47 Kb). Yeast artificial chromosome (YAC) are used in yeast. YACs have an origin to support replication, a centromere to ensure proper segregation, and telomeres to afford stability. Bacterial artifical chromosomes (BAC) are based on the E. coli F plasmid. BACs can carry ~300 kb of DNA. They are more stable than YACs. YACs. Elasri USM BSC476/576 Shuttle Vectors Cloning vector can be engineered to replicate in more than one host. pYac2 is can replicate in bacteria (E. coli) and yeast (S. cerevisiae). coli) (S. cerevisiae) pYac2 has bacterial features that include an origin of replication and antibiotic resistance gene. It also has yeast features that include an origin, centromere, two centromere, selectable markers, and telomeres. Elasri USM BSC476/576 Restriction Enzymes DNA can be cleaved with restriction enzymes in a precise manner. There numerous restriction enzymes that have been isolated and characterized. Each restriction enzyme cleaves a specific target sequence (usually 4-6 bp in 4length). The sites of cleavage on the two strands may generate 5 protruding ends, 3 protruding ends, or blunt ends. Elasri USM BSC476/576 Recombinant Plasmid The insert DNA and the vector are cleaved with the same restriction enzyme(s). enzyme(s). The two molecules of DNA are then isolated and ligated together to make a recombinant plasmid. Selection using antibiotic genes are used to eliminate cells that are not that carrying the vector. Antibiotic genes can also be used to distinguish between recombinant recombinant plasmids (carrying the donor DNA) and the original vector. Example: Bacteria carrying a cloning vector can be selected by resistance to an antibiotic marker carried by the vector (ampicillin or neomycin). (ampicillin Bacteria carrying a recombinant cloning vector with an insertion of donor DNA can be distinguished by loss of activity of the vector gene (ampicillin) into which the insertion was made. ampicillin) Selective Markers Elasri USM BSC476/576 Elasri USM BSC476/576 Reporter Genes Reporter gene are used to examine the regulation of a gene. The promoter of the gene of interest is fused with a promoterless reporter gene whose gene product can be easily assayed. When the promoter is active the reporter gene is expressed allowing easy monitoring. Example: Expression of a lacZ gene can be followed in the mouse by staining for galactosidase (addition of a chromogenic sbstrate that turns blue after reaction with -galactosidase). galactosidase). lacZ was expressed under the control of a promoter of a mouse gene that is expressed in the nervous system. Elasri USM BSC476/576 Transfection Transfection of eukaryotic cells is the acquisition of new genetic markers by incorporation of added DNA. The transfected unit is unstable unless it becomes integrated into a host chromosome. Transient transfectants have foreign DNA in an unstable, i.e. extrachromosomal, extrachromosomal, form. Stable transfectants have foreign DNA integrated into a random chromosomal site. Transfected DNA can be expressed. Elasri USM BSC476/576 Transfection into the Germline DNA that is injected into animal eggs can integrate into the genome. Transgenic animals are created by introducing DNA prepared in test tubes into the germline. germline. The DNA may be inserted into the genome or exist in an extrachromosomal structure. Elasri USM BSC476/576 Transgenic Animals Transgenic mouse created by injection of an active rat growth hormone gene is twice the size of a normal mouse. The transgenic mouse had growth hormone levels several hundred times greater than normal. These techniques can be used to cure diseases by replacing defective genes with functional ones. Elasri USM BSC476/576 Gene Targeting Gene targeting allows genes to be replaced or knocked out An endogenous gene can be replaced by a transfected gene using homologous recombination. A gene knockout is a process in which a gene function is eliminated, usually by replacing most of the coding sequence with a selectable marker in vitro and transferring the altered gene to the genome by homologous recombination. A gene knock-in is a process similar to knocka knockout, but more subtle mutations are made, such as nucleotide substitutions or the addition of epitope tags. 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