Lecture 2: Techniques in Biotechnology PDF

Lesson 2: Techniques in Biotechnology METHODS OF GENETIC TRANSFORMATION Presented by: Trishia Jade C. Acilo Instructor I Department of Plant Breeding and Genetics Visayas State University Visca, Baybay City, Leyte Gene Transfer/Genetic Transformation This involves transfer of DNA sequences from...

Lesson 2: Techniques in Biotechnology METHODS OF GENETIC TRANSFORMATION Presented by: Trishia Jade C. Acilo Instructor I Department of Plant Breeding and Genetics Visayas State University Visca, Baybay City, Leyte Gene Transfer/Genetic Transformation This involves transfer of DNA sequences from one organism to another The transferred DNA has to be incorporated into the DNA in the nucleus or mitochondrion or plastid, depending on your target compartment The DNA sequences to be transferred may or may not be foreign (i.e. coming from another species) This is usually done when crossbreeding is not possible (i.e. because of sterility of the species or absence of desired gene within the species) There are a variety of methods of transferring genes (also referred to as transformation or transgenesis) available to plants and animals. The methods generally require that the recipient cell be competent, i.e. must have the ability to take up DNA to be transformed  http://upload.wikimedia.org/wikipedia/commons/thumb/8/80/Breeding_transgenesis_cisgenesis.svg/700px-Breeding_transgenesis_cisgenesis.svg.png https://en.wikipedia.org/wiki/Cisgenesis Comparison of conventional breeding, transgenesis and cisgenesis in the transfer of desired gene/s. Comparing cisgenesis and transgenesis https://www.slideshare.net/mohammedsami31508/plant-transformation-methods  A.turmefaciens Indirect:Agrobacterium Method Crown gall This method relies on the soil bacterium Agrobacterium tumefaciens to transfer the gene of interest into the genome of plant cells Agrobacterium tumefaciens is a soil-borne bacterium Causes Crown gall disease Agrobacterium is a natural genetic engineer This bacterium has a natural ability to transfer the so-called T-DNA in its Ti plasmid into plant cells By replacing its T-DNA with desired gene/s, Agrobacterium is able to transfer the desired genes into the genome of target plants Agrobacterium Method The Ti-plasmid Ti-plasmid from different strains of A. turmefaciens has some features in common: One or more regions of T-DNA a vir-region an origin of replication a region enabling conjugative transfer genes for catabolism of opines Agrobacterium is a natural genetic engineer (https://bioone.org/journals/the-arabidopsis- book/volume-2017/issue- 15/tab.0186/iAgrobacteriumi-Mediated-Plant- Transformation-Biology-and- Applications/10.1199/tab.0186.full) Two genetic components essential for Agrobacterium transformation T-DNA Vir Region (virA, virB, virC, virD, virE, virF, and virG http://images.google.com/imgres?imgurl=http://www.mcdb.lsa.umich.edu/labs/ Agrobacterium-mediated transformation Gene Gun or Biolistic Method Gene gun or particle delivery system is a device used to deliver exogenous DNA, RNA or protein to cells By coating a heavy metal (e.g. tungsten or gold) microprojectiles with the gene of interest and firing these into cells, the cells can be transformed. The DNA construct to be delivered into cells by this method should be fully functional, i.e. it should contain the promoter and terminator sequences, the gene of interest and a selectable marker gene and sometimes also a reporter gene How gene gun works? Target material in Plants: callus of the undifferentiated cells of the plant http://images.google.com/imgres?imgurl=http://www.mcdb.lsa.umich.edu/labs/ Standard Gene gun Transformation by particle bombardment  Electroporation Method Also called electrotransfer, this method can be used to transform plant protoplasts The device used for electroporation is an electroporator The cell suspension is pipetted into a glass or plastic cuvette which has two aluminium electrodes on its sides The electroporator creates an electrostatic field in the cell suspension An electroporator and some Electrical shocks causes holes in the cuvettes membranes. The foreign DNA fragments will enter through the holes created and will go into the cytoplasm and then to nucleus Electroporation Method Upon the introduction of an electrical pulse to the cell suspension, temporary pores form on the cell membrane and DNA or other desired molecules can enter the cell The DNA may then enter the nucleus and later become incorporated into the nuclear DNA The protoplast are then cultured in vitro to produce calli and later, whole plants https://blog.universalmedicalinc.com/what-is-electroportation/ Whisker-mediated transformation https://www.researchgate.net/figure/Scheme-of-whisker-mediated-transformation-of-soybean_fig2_241753945 Chemofusion- chemical method ✓ involves treatment of protoplast and other cells/tissues with polyethylene glycol (PEG), calcium phosphate and etc. Genetic Transfer in Animals In animals, this term transformation is replaced by the term 'transfection' because the term transformation is used in animals to describe phenotypic alteration in cells. Transfection or gene transfer in animals can be carried out at the cellular level. Transfected cells may be used for a variety of purposes; (i) production of chemicals and pharmaceutical drugs; (ii) study of structure and function of genes and (iii) production of transgenic animals The transfection methods may employ the fertilized eggs/embryos or the cultured cells. Genetic Transfer in Animals I. Transfer of genes to fertilized eggs or embryos. A. Transfer of whole nuclei - the egg cells are treated with cytochalasin- B and subjected to centrifugation causing enucleation (nucleus is removed). Incubation of desirable karyoplasts with these enucleated eggs leads to the transfer of whole nuclei in presence of polyethylene glycol (PEG). B. Transfer of whole chromosomes- the chromosomes are first isolated by hypotonic lysis and may be fractionated using density flow cytophotometry. Individual chromosomes or fragments isolated are then incubated with whole cells (eggs) for incorporation of chromosomes into nuclei; Genetic Transfer in I. Transfer of genes to fertilized eggs or Animals embryos. C. Microinjection of DNA segments - the most used method. Several hundred copies of the DNA segment are utilized and after stable integration, the eggs are used for getting transgenic animals. - provides direct nuclear delivery of DNA ensuring that the DNA is delivered intact. Microinjection of foreign DNA into a fertilized egg for transfer of a gene leading to the production of transgenic animals. Genetic Transfer in Animals II. Transfer of genes to cultured cells. Purposes: gene therapy production of valuable proteins production of transgenic plants. (i) coprecipitation of DNA with calcium phosphate (ii) electroporation (a technique, where cell membrane is made more permeable by electric shocks (iii) use of gold or tungsten particles coated with DNA and accelerated to a high speed using a particle gun (v) microinjection of DNA using micro-manipulator and vi) use of liposomes or sphaeroplasts, which are vesicles carrying DNA Transfection of stem cells and injection of derived stem cells into blastocyst, leading to the production of transgenic organism Methods of plant transformation: I. INDIRECT METHOD: Agrobacterium, or virus- mediated gene transfer. II. DIRECT METHODS : Particle gun/biolistic/ballistic method of DNA delivery. Chemical method: Polyethylene glycol (PEG)/protoplast fusion. Microinjection Electroporation Lipofection- Liposome mediated gene transfer. Fiber mediated DNA deliver/whisker Lesson 2: Techniques in Biotechnology METHODS OF GENETIC TRANSFORMATION Presented by: John Raphael P. Camba Part-time Instructor Department of Plant Breeding and Genetics Visayas State University Visca, Baybay City, Leyte