Genetic Engineering Application of techniques of molecular cloning and transformation

Applications _ Improving plant technology: Genetically modified (GM) plants that are bacteria or pest resistant. Oral vaccination in plants. _Manufacture of human insulin using modified bacteria. _Production of experimental mice (eg. Oncomouse )for research. _Manufacture of growth hormones. _Manufacture of vaccine for hepatitis B

Disadvantages # Introduction of genetically engineered organisms into environment can disturb natural ecosystem. # Human consumption of some genetically modified food may be dangerous.

Methodology for GE Isolation of gene of interest. Isolation of gene of interest. Insertion of the gene into a vector. Transformation of the cells of the organism that has to be modified. Test for isolation of GMO.

Transformation It is the process by which the vector with inserted gene of interest is introduced into the cell of target organism. It can be done by following methods: _Transduction _Transduction _ Transfection _ Transfection

Transduction The vector containing gene of interest is inserted into a virus. This virus is used to incorporate the target DNA into the genome of a cell.

Transfection Introducing Naked DNA into cultured cells. It can be done by following methods: -Spontaneous uptake -Spontaneous uptake -Electroporation -Electroporation -Microinjection -Microinjection

Spontaneous uptake Cells are suspended in presence of a fine calcium precipitate of DNA. 1 out of 100,000 – 1000,000 cells take up the DNA and incorporates it into its chromosome. These cells are called transfected cells.

Electroporation Cells are incubated with DNA in special vials that contain electrodes that deliver a brief electric shock. Application of this electric current causes the plasma membrane to become transiently permeable to DNA molecules. Some DNA enters cell and is incorporated with the cells genome.

Microinjection In this method the DNA is injected directly into the cell nucleus. Nucleus of the oocytes and eggs are suited for this approach.

GE and research Loss of function experiment- the organism is engineered to lack the activity of one or more gene. This allows the experimenter to analyze the defects caused by this mutation and to dtudy the function of this gene. Eg. Knockout mouse.

GE and research Gain of function experiment- it is counterpart of knockout. Extra copies of gene of interest are inserted in the DNA construct.

GE and research Tracking experiments-these experiments are done to gain information about localization and interaction of the desired protein. For this the gene of interest is fused with gene for producing flurescence. The protein translated by the gene will be fluorescent. OR By adding a small sequence which will serve as binding site for monoclonal antibodies.

GE and research Expression studies- to discover where and when specific proteins are produced. In this the promoter region of the gene of interest is introduced into an organism whose protein coding region for that gene has fusion of fluroscence gene. Thus the time and place where a particular protein is produced is observed.

Gene Therapy the ability to replace known disease genes with normal copies in afflicted humans is the ultimate goal of gene therapy. Human gene therapy protocols aim to introduce correcting copies of disease genes into somatic cells of the affected individual. Human gene therapy protocols aim to introduce correcting copies of disease genes into somatic cells of the affected individual. This prevents transmission through the germ line, thereby, avoiding many of the ethical issues of transgenesis.

. The most common techniques utilized in gene therapy studies is the introduction of the corrected gene into bone marrow cells, skin fibroblasts or hepatocytes. The vectors most commonly utilized are derived from retroviruses. The advantage of retroviral-based vector systems is that expression occurs in most cell type.

Human Disorders Treated in Cultured Cells by Gene Therapy Disorder Affected Gene SCID* - Adenosine deaminase SCID - Purine nucleoside pyrophosphate Phenylketonuria-Phenylalaninehydroxylase β-Thalassemia-β-Globin Phenylketonuria β-Thalassemia Phenylketonuria β-Thalassemia Hemophilia BHemophilia B-Factor IX Hemophilia B * Severe combined immunodeficiency disease