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Submitted By:. Bikash Karki Shrawan Shah Submitted TO Anup Sharma IVth Semester Bsc. Biotechnology SANN college Dines S. Pujara

Presentation overview

# Introduction To GMO # Transgenic Animal # History # Applications Of Transgenic Animals #Methods To Produce GMO # Knockout Mouse Project # Application of KOMP # Myths And Ethics # Conclusion # References

WHAT ARE GMO or TRANSGENIC ANIMALS ? A genetically modified organism (GMO) or genetically engineered organism (GEO) is an organism whose genetic material has been altered using genetic engineering techniques.These techniques are generally known as recombinant DNA technology. With this technology, DNA molecules from different sources are combined into one molecule to create a new set of genes. This DNA is then transferred into an organism and causes the organism to acquire modified or novel traits. Transgenic Animals A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to a structural gene, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host and to be expressed correctly by the cells of the host.

History of transgenic animal production:

Discovery of DNA and the creation of the first recombinant bacteria in 1973, i.e., E .coli expressing a salmonella gene 1970's, first transgenic mice via viral infection, but not germline transmission. 1980's, first transgenic mice via microinjection, the most popular technique 1985, first transgenic rabbits, sheep, pigs and cattle 80-90, commercial transgenic services, via transgenic facility ‡ 1990's, transgenic farm animal companies as bioreactors and organ donors

Applications Of GMO¶s
IN GENERAL Includes transgenic animals [mice, fish (aquaculture)], transgenic plants, various microbes, such as fungi and bacteria.

In research that addresses fundamental or applied questions in biology or medicine, for the production of pharmaceuticals and industrial enzymes applications aimed at improving human health (e.g., gene therapy) agriculture (e.g., golden rice).

The term ³GMO" does not always imply targeted insertions of genes from one into another species For example, a gene from a jellyfish, encoding a fluorescent protein GFP, can be physically linked and coexpressed with mammalian genes to identify the location of the protein.

Transgenic microbes
Medical  to produce the protein insulin, in treatment of treat diabetes  to produce clotting factors to treat haemophilia  human growth hormone to treat various forms of dwarfism  genetically modified viruses allow gene therapy , is being developed for a treatment of incurable diseases, such as cystic fibrosis, sickle cell anemia and muscular dystrophy  is also used in some soils to facilitate crop growth, and can also produce chemicals which are toxic to crop pests WHY TRANSGENIC MICROBES??? Because, these recombinant proteins are much safer than the products they replaced, since the older products were purified from cadavers and could transmit diseases.

Transgenic Plants
resistance to pests, herbicides (eg. glufosinate or glyphosate and events producing the Bt toxin) or harsh environmental conditions improved shelf life increased nutritional value Whenever GM plants are grown on open fields without forms of containment, there is the possibility that there could be associated environmental risks. Therefore, most countries require biosafety studies prior to the approval of a new GM plant event, usually followed by a monitoring programme to detect environmental impacts.

Results of insect infestation on Bt (right) and non-Bt (left) cotton bolls. Source: USDA

Effect of the herbicide bromoxynil on tobacco plants transformed with a bacterial gene whose product breaks down bromoxynil (top row) and control plants (bottom row). "Spray blank" plants were treated with the same spray mixture as the others except the bromoxynil was left out. (Courtesy of Calgene, Davis, CA.)

Transgenic Animals: Historical Background
During the 1970s, the first chimeric mice were produced (Brinster, 1974) Cells of two different embryos of different strains were combined together at an early stage of development (eight cells) to form a single embryo that subsequently developed into a chimeric adult, exhibiting characteristics of each strain. DNA microinjection, (Gordon and Ruddle ),1981 the first technique being proved successful in mammals, was first applied to mice and then to various other species such as rats, rabbits, sheep, pigs, birds, and fish the term transgenic was first used by J.W. Gordon and F.H. Ruddle (1981) Two other main techniques were then developed those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986).

Methods of creation of transgenic animals

Direct microinjection Virus mediated gene transfer Nuclear transfers Sperm-mediated gene transfer Artificial chromosomes for gene transfer Embryonic stem cells (KNOCKOUT MOUSE)

Direct microinjection
‡ inject DNA molecules (transgenes) directly into male pronucleus ‡ manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother ‡ most popular technology, commercial available ‡ the success rates range from 10-30% depending on skills and constructs ‡ the insertion of DNA is a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression ‡ the efficiency is not related to the copies of transgenes injected ‡ initial investment is high, a minimum of $100K to start

Virus Mediated Gene Transfer 

gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid  Retroviruses are commonly used as vectors  killed virus is replication defective  the virus gene is replaced with transgene  the transgene is delivered to the host cell by transfection (gene therapy)  can be used to transfect a wide range of cells, e.g., ES cells

Nuclear transfer
‡ creation of Dolly ‡ somatic cells be transfected, or genetically altered prior to NT ‡ 100% efficiency of any progeny ‡ abnormal development

WHAT IS DOLLY????

The Embryonic Stem Cell Method
Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell. These cells are incorporated into an embryo at the blastocyst stage of development. Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. Structural gene of desire is inserted OR knocked out via DNA-Recombination. Successfully transformed cells are selected and injected into inner cell mass of mouse blastocyst. Embryo transfer Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive. Transfer the embryos into her uterus.

What is a knockout mouse? A knockout mouse is a genetically engineered mouse in which one or more genes have been turned off through a gene knockout. Knockout mice are important animal models for studying the role of genes which have been sequenced, but have unknown functions. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or condition, researchers can infer its probable function. Mice are currently the most closely related laboratory animal species to humans, for which the knockout technique can easily be applied.

The Nobel Prize in Physiology or Medicine 2007 "for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells³ is awarded to..

Mario R. Capecchi Sir Martin J. Evans Oliver Smithies
USA University of Utah United Kingdome Cardiff University USA University Of North Carolina

The first knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver Smithies in 1989, for which they were awarded the Nobel Prize for Medicine in 2007.

PRODUCTION OF KNOCKOUT MICE.
CONCEPT OF KNOCKOUT MOUSE PRODUCTION BEGINS AFTER THE HARVESTING OF EMBRYONIC STEM CELLS FROM EARLY-STAGE MOUSE ENBRYOS 4 DAYS AFTER FERTILIZATION.

WHY ALWAYS EMBRYONIC STEM CELLS, NOT OTHERS ?
TOTIPOTENCY: Capable to differentiate into nearly any type of adult cell GERMLINE EFFECT: If a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse. VIABILITY: ES cells grown in the lab can be used to make knockout mice as long as 10 years after they were harvested.

Harvesting of ES cells

Depending upon methods of insertion of artificial DNA into the chromosome of ES cells nuclei there are TWO METHODS TO PRODUCE KNOCKOUT MOUSE. Both methods are carried out in vitro.

o Homologous recombination OR Gene targeting o Gene trapping
introduction of an artificial piece of DNA sharing identical, or homologous, sequence to the target gene. homologous sequence flanks the existing gene's DNA sequence both upstream and downstream of the gene's location on the chromosome. cell's own nuclear machinery automatically recognizes the identical stretches of sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA. Because the artificial DNA is inactive, bearing only a genetic tag, or "reporter gene," designed for use in tracking, the swap eliminates, or "knocks out," the function of the existing gene. appearance of change in any phenotypic sign implies the function of knocked out gene.

BLASTOCYST

herpes virus thymidine kinase (tk) gene

Targeted ES cells are inserted in to blastocyst of foster mother

Chimeric F1 mice

Chimeric male is crossed with normal female.

Targeted (homozygous) & normal mice. (F2)

Gene Trapping OR Random Insertion

AND«««.
For both gene targeting and gene trapping, a modified viral vector or a linear fragment of bacterial DNA is used to insert the artificial DNA into ES cell. After insertion, the genetically altered ES cells are grown in a lab dish for several days and injected into early-stage mouse embryos. The embryos are implanted into the uterus of a female mouse and allowed to develop into mouse pups. The resulting mouse pups are not complete knocked out, that is with both normal & altered ES cells. These are crossbreed to produce lines of mice in which both copies of the gene are knocked out in all tissues, called homozygous knockouts.

Altered or mutated phenotype (appearance, biochemical characteristics, behavior etc.) of the pups give clue about the gene¶s normal function.

Which Production Method Is Best? 
Gene trapping do not need target DNA to be sequenced.  Single vector can be used to knock out various genes. BUT, L Not every successful insertion of artificial DNA into a gene leads to a loss of function. L Must spend considerable time conducting tests to identify ES cells in which gene actually have been knocked out .

So Gene Targeting is widely used.

Some NGOs are still working on further development of gene targeting and to reduce biological ethics of knockout mouse and transgenic animal.

KNOCKOUT MOUSE PROJECT DATA COORINATION CENTRE (KOMP DCC), centered at The Jackson Laboratory in Bar Harbor Maine EUROPIAN CONDITIONAL MOUSE MUTAGENESIS PROGRAMS (EUCOMM) NORTH AMERICAN CONDITIONAL MOUSE MUTAGENESIS PROGRAMS (NACMM)

Significance of gene targeting for physiology and medicine

Knockout mice gives information that can be used to better understand how a similar gene may cause or contribute to disease in humans. Atherosclerosis

Inherited Heart Disease

Monogenic Disease
Lesch-Nyhan syndrome Cystic fibrosis inherited heart diseases

Complex Disease
Essential hypertension Atherosclerosis

Cancer Other diseases
Obesity, Diabetes, Arthritis, Substance abuse, Anxiety, Aging and Parkinson's disease.

Gene Targeting In Disease Diagnosis And Study

Lesch-Nyhan syndrome, due to mutation in HPRT (phosphoribosyltransferase ) gene cause a defective nucleotide metabolism in human. Similar gene adenine phosphoribosyltransferase (APRT) for purine salvage in mouse was observed, cause of neuropathological & change in behavioral feature.

Cystic fibrosis, Defective chloride transport through cAMP-activated chloride channel in mouse due to knocking out of cystic fibrosis transmembrane conductance regulator (CFTR) gene. The similar phenotypes is observed in human as in mice. Essential Hypertension, 10 genes are responsible for altering blood pressure. Angiotensinogen (AGT) gene polymorphism is associated with essential hypertension. Targeting of Angiotensin-converting enzyme (ACE) coding gene in mouse is observed to be effective in reduction of hypertension. Similarly renin-angiotensin system is applied in human for hypertension. Cancer, Protooncogenes, tumor suppressor genes, angiogenetic factors targeted in mice to know about the induction and spreading of tumours & their role in the formation of tumors. These targeted mouse being solid support to study of cancer in human.

How do transgenic animals contribute to human welfare????

Agricultural Applications
Breeding; Quality Disease Resistance

Medical Application Xenotransplantation
Nutritional Supplements and Pharmaceuticals Human Gene Therapy

Industrial Application
Nexia Biotechnologies in Canada transformed spider gene in goat & it began to secrete tiny silk strands from their body. By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical microsutures, and tennis racket strings.

Ethical concerns surrounding transgenesis

Transgenic animals raise several particular moral issues
C Should there be universal protocols for transgenesis? C Is human welfare the only consideration? What about the welfare of other life forms? C Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering? C Will transgenic animals radically change the direction of evolution, which may result in drastic consequences for nature and humans alike? C Should patents be allowed on transgenic animals, which may hamper the free exchange of scientific research? C Organ rejection!! C Fail to give observable change. Religious views of transgenic animals: God laid down the structure of creation and any tampering with it is sinful

CONCLUSION
Interestingly, the creation of transgenic animals has resulted in a shift in the use of laboratory animals, from the use of higher-order species such as dogs to lower-order species such as microbes, and has decreased the number of animals used in such experimentation, especially in the development of disease models. This is certainly a good turn of events since transgenic technology holds great potential in many fields, including agriculture, medicine, and industry.

THANK YOU !

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