REKAYASA GENETIKA (GENETIC ENGINEERING)Etty Widayanti, SSi. MBiotech.Bagian Anatomi Sub Bagian BiologiFak. Kedokteran Univ. YARSINov 2009

The alteration of an organisms genetic, or hereditary, material to eliminate undesirable characteristics or to produce desirable new ones.The simple addition, deletion, or manipulation of a single trait in an organism to create adesired change.

Definition:What is genetic engineering?

Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.

Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

Introduction of genes from one species of organism intoorganism of a different species (transgenic organism) Cloning a gene to determine its function Change the phenotype of an organism to improve a desired trait

Traditional genetic manipulationSelective breeding to improve nutritional value of plants too slow

Genetic engineering by molecular techniques Allows to create larger genetic changes at a much more rapid pace Production of large amounts of pharmaceuticalsWhy do scientists genetically engineer organisms?

Genetic Engineering Techniques

Selective BreedingPlants or animals with most desirable characteristics used for further breedingHybridization (Crossbreeding)Combining different strains of a species to combine the best characteristics of both. (female horse + male donkey = Mule)Recombinant DNA TechnologiesTransformation

Basic steps in genetic engineeringIsolate the geneInsert it in a host using a vectorProduce as many copies of the host as possibleSeparate and purify the product of the geneExtraction of desired of gene product

Fluorescent proteins are a useful tool in biotechnologyProvide convenient markers for gene expression and protein targeting in living cells and organisms.

Most widely used is green fluorescent protein (GFP) first isolated from the jellyfish Aequorea victoria, can be attached to virtually any protein of interest and still fold into a fluorescent molecule.

Can be used to localize previously uncharacterized proteins or to visualize and track known proteins to further understand cellular events.

Can be used for process development

Before 1980sInsulin extracted from the glands of cows and pigs.

Worked to lower blood sugar, but seen by the immune system as "foreign" and induced an antibody response, thus reducing the effect and ultimately requiring higher doses.

Converted pig insulin into human insulin by removing the one amino acid that distinguishes them and replacing it with the human version ExpensiveThe Power of Biotech.

Insert the human gene for insulin into E. coli and grow recombinant human insulin in culture tanks.

E. coli is able to manufacture a fully-functional molecule (trade name = Humulin). Yeast is also used (trade name = Novolin). The Power of Biotech.

Insulin (diabetics) Factor VIII (males w/hemophilia A) Factor IX (hemophilia B) Human growth hormone Erythropoietin (anemia) Angiostatin/endostatin(anti-cancer drugs) leptinRecombinant DNA products being used in human therapy

mouse with human ear

Regenerative Medicine with Stem Cells

http://www.news.wisc.edu/packages/stemcells/illustration.html Images depict stem cell research at the University of Wisconsin Madison.

http://www.kumc.edu/stemcell/early.html Reprinted with permission from the University of Kansas Medical Center.

Genetically Engineering HumansBone marrow supplies stem cells

Successful replacement of gene for enzyme needed for lymphocyte development

Health and Medicine- affecting health care & medical industry.- alternating growth w/hormonesreplacing organs are common.- materials maybe rejected by organism unless hormones are offered.

Difficulties in GeneticallyEngineering HumansInserting gene in correct cellsInserting gene so it is expressed correctly Orientation RegulationControlling virus vectorEthical issues

Transgenic AnimalsKnock-out and transgenic mice: used to study immune system and genetic diseasesPigs: blood clotting Factor VIII, organs for transplantationOthers: Human IL-2 (cancer), albumin (blood volume), growth hormone, tPA (dissolves clots)

Some considerations: - Ti plasmid is 200 kb - too big for easy cloning need to remove tumor-inducing genes from T-DNA

Genetic engineering of the Ti plasmid

Genetically Modified OrganismsHerbicide-resistant plantsBt cotton/corn (toxin gene from Bacillus thuringiensis that kills insects)Flavr-Savr tomatoesGolden rice (beta-carotene)Plant-based vaccines

Genetic engineering of Bt-resistant corn (Bt-corn) Bt-transgenic plants are resistant to insects, no need for sprayinginsecticide Clone Bt toxin gene from bacteria and express in plants

Examples of applications of genetic engineering in agriculture

Golden Rice containing genes for production of vitamin A Rice is a major staple food for half of the worlds population. Rice grains (endosperm) lack several essential nutrients, including vitamin A and its precursors (-carotene). Vitamin A deficiency causes blindness and weakness of theimmune system (effects ~ 400 million people worldwide).

Golden Rice Generate transgenic rice containing entire new metabolic pathway

-carotene pathway includes 4 enzymesGeranylgeranyl diphosphate (in immature rice endosperm)-carotene Vitamin A

Pharmacology-preparation, use, and affect of drugs tied to health and medicine potential production of drugs is greathormone production-natural in endocrinesystem of mammalian bodyPharmaceutical Products-bacteria engineered to produce hormone-fermentation known as bioprocessing

DNAproteindrugs are so complex they can only be synthesized in a living system

Industry and Environment more efficient use of scarce minerals

greater efficiency in mining

major concern-waste management

biodegrade a number of waste products sewage and petroleum products

Industry and Environment

ReferencesCampbell, N.A., Reece, J.B. and Mitchell, L.G. 2004. Biologi. Jilid ke-3. Ed ke-5. Penerbit Erlangga, Jakarta.Pinkert, C.A. 2002. Transgenic animal technology: A laboratory handbook. 2nd ed. Academic Press, California.Schmid, R.D. 2003. Pocket guide to biotechnology and genetic engineering. Wiley-VCH, Weinheim.Yuwono, T. 2006. Bioteknologi pertanian. Gadjah Mada University Press, Yogyakarta

Gene technology used in same way as genetic engineering, genetic fingerprinting no manipulation of DNAGenetic engineering is a very young discipline, and is only possible due to the development of techniques from the 1960s onwards. These techniques have been made possible from our greater understanding of DNA and how it functions following the discovery of its structure by Watson and Crick in 1953. Although the final goal of genetic engineering is usually the expression of a gene in a host, in fact most of the techniques and time in genetic engineering are spent isolating a gene and then cloning it. This table lists the techniques that we'll look at in detail.

Need to explain some key terms first before we talk about how we would isolate the gene and clone it