knock out technology By Biksh

8/9/2019 knock out technology By Biksh

1/32


2/32

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


3/32

A genetically modifiedorganism (GMO) orgenetically engineered

organism (GEO) is an organism whose genetic material has been alteredusing genetic engineering techniques.These techniques are generally knownas recombinant DNA technology. With this technology, DNA molecules fromdifferent sources are combined into one molecule to create a new set ofgenes. This DNA is then transferred into an organism and causes theorganism to acquire modified or novel traits.

Transgenic Animals

A transgenic animal is one that carries a foreign gene that has beendeliberately inserted into its genome. The foreign gene is constructed usingrecombinant DNA methodology. In addition to a structural gene, the DNAusually includes other sequences to enable it

WHAT ARE GMO or TRANSGENIC ANIMALS ?

to be incorporated into the DNA of the host and to be expressed correctly by the cells of the host.


4/32

1970's,firsttransgenic mice via viral infection, but notgermline

transmission.

1980's,firsttransgenic mice via microinjection,the most popular

technique 1985,firsttransgenic rabbits,sheep, pigs andcattle

80-90,commercial transgenicservices, via transgenicfacility

1990's,transgenicfarm animal companies as bioreactors andorgan

donors

History oftransgenic animal production:

Discovery ofDNA andthecreation ofthefirst recombinant

bacteria in 1973,i.e.,E .coliexpressing a salmonella gene


5/32

Applications OfGMOs

Includestransgenic animals [mice,fish (aquaculture)],transgenic plants,various microbes,such asfungi and bacteria.

In research that addresses fundamental or applied questions inbiology or medicine, for the production ofpharmaceuticals and

industrial enzymes

applications aimed at improving human health (e.g., gene therapy)

agriculture (e.g., golden rice).

IN GENERAL

The term GMO" 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 co-

expressed with mammalian genes to identify the location of the protein.


6/32

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 ofdwarfism

genetically modified viruses allow gene therapy , is being developed for

a treatment of incurable diseases, such as cystic fibrosis, sickle cellanemia and muscular dystrophy

is also used in some soils to facilitate crop growth, and can also producechemicals which are toxic to crop pests

WHY TRANSGENIC MICROBES???

Because, these recombinant proteins are much safer than the products theyreplaced, since the older products were purified from cadavers and couldtransmit diseases.


7/32

Transgenic Plants

resistance to pests, herbicides (eg. glufosinate orglyphosate 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 anew GM plant event, usually followed by a monitoring programme to detectenvironmental impacts.

Resultsofinsectinfestation on Bt

(right) and non-Bt (left) cotton

bolls. Source: USDA


8/32

Effectofthe herbicide bromoxynil on tobacco plantstransformed with a bacterial

gene whose product breaksdown bromoxynil (top row) andcontrol plants

(bottom row). "Spray blank" plants weretreated with thesamespray mixture astheothersexceptthe bromoxynil was leftout. (Courtesy ofCalgene, Davis, CA.)


9/32

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 anearly stage of development (eight cells) to form a single embryo that subsequentlydeveloped 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).


10/32

Methods of creation of transgenic animals

Direct microinjection

Virus mediatedgenetransferNucleartransfers

Sperm-mediatedgenetransfer

Artificial chromosomesforgenetransfer

Embryonicstem cells

(KNOCKOUT MOUSE)


11/32

Direct microinjection inject DNA molecules (transgenes) directly into male pronucleus manipulated fertilized ovum is transferred into the oviduct of a recipient female, orfoster 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 theintroduced gene will not insert itself into a site on the host DNA that will permit itsexpression the efficiency is not related to the copies of transgenes injected initial investment is high, a minimum of $100K to start


12/32


13/32

Nucleartransfer

creation ofDolly

somaticcells betransfected,orgenetically altered priorto NT 100%efficiency ofany progeny

abnormal development

WHAT IS DOLLY????


14/32


15/32


16/32


17/32

Whatis 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 havebeen sequenced, but have unknown functions. By causing a specific gene to beinactive in the mouse, and observing any differences from normal behaviour orcondition, researchers can infer its probable function.

Mice are currently the most closely related laboratory animal species to humans, forwhich the knockout technique can easily be applied.


18/32

The Nobel Prizein Physiology or Medicine 2007

"for their discoveries of principles for introducing specific genemodifications in mice by the use of embryonic stem cellsis awarded to..

Mario R. CapecchiUSA

University of Utah

Sir Martin J. EvansUnited KingdomeCardiff University

Oliver SmithiesUSAUniversity Of North Carolina

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


19/32

PRODUCTION OF KNOCKOUT MICE.

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 beobserved in any tissue in an adult mouse.

VIABILITY: ES cells grown in the lab can be used to make knockout mice as longas 10 years after they were harvested.

CONCEPT OF KNOCKOUT MOUSE PRODUCTION BEGINS AFTER THE HARVESTING

OF EMBRYONIC STEM CELLS FROM EARLY-STAGE MOUSE ENBRYOS 4 DAYSAFTER FERTILIZATION.

WHY ALWAYS EMBRYONIC STEM CELLS, NOT OTHERS ?

Harvesting of ES cells


20/32

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

o Homologous recombinationOR 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 anddownstream of the gene's location on the chromosome.

cell's own nuclear machinery automatically recognizes the identical stretches of sequenceand 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 existinggene.

appearance of change in any phenotypic sign implies the function of knocked out gene.


21/32


22/32

herpes virus thymidine kinase (tk) gene

BLASTOCYST


23/32

Targeted ES cells are

insertedin to

blastocystoffoster

mother

ChimericF1 mice

Chimeric maleiscrossed

with normal female.

Targeted (homozygous) & normal mice. (F2)


24/32

Gene Trapping OR Random Insertion


25/32

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 severaldays and injected into early-stage mouse embryos. The embryos are implanted into the uterus of a female mouse and allowed todevelop 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 copiesof the gene are knocked out in all tissues, called homozygous knockouts.

AND.

Altered or mutated phenotype (appearance, biochemical characteristics, behavioretc.) of the pups give clue about the genes normal function.


26/32

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 agene leads to a loss of function.

L Must spend considerable time conducting tests toidentify ES cells in which gene actually have been

knocked out .

So Gene Targetingis widely used.


27/32

KNOCKOUT MOUSE PROJECT DATA COORINATION CENTRE (KOMP DCC),centered at

The Jackson Laboratory in BarHarbor Maine

EUROPIAN CONDITIONAL MOUSE MUTAGENESIS PROGRAMS (EUCOMM)

NORTH AMERICAN CONDITIONAL MOUSE MUTAGENESIS PROGRAMS (NACMM)

Some NGOs arestill workingon furtherdevelopmentofgenetargeting andto

reduce biological ethicsofknockout mouse andtransgenic animal.


28/32

Monogenic Disease

Lesch-Nyhan syndrome

Cysticfibrosis

inherited heartdiseases

Cancer

Otherdiseases

Obesity, Diabetes, Arthritis, Substance abuse, Anxiety, Aging andParkinson'sdisease.

Significance of gene targeting for physiology and medicine

Knockout micegivesinformation thatcan be usedto better understand how a similar

gene may causeorcontributetodiseasein humans.

Complex Disease

Essential hypertension

Atherosclerosis

Atherosclerosis

Inherited Heart Disease


29/32

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

Cysticfibrosis, Defective chloride transport through cAMP-activated chloride channel in mousedue to knocking out ofcysticfibrosistransmembraneconductance 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. Targetingof 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 forhypertension.

Cancer, Protooncogenes, tumor suppressor genes, angiogenetic factors targeted in mice toknow 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.

Gene Targeting In Disease Diagnosis And Study


30/32

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 tinysilk strands from their body. By extracting polymer strands from the milk and weaving theminto thread, the scientists can create a light, tough, flexible material that could be used in suchapplications as military uniforms, medical microsutures, and tennis racket strings.


31/32

Ethical concernssurroundingtransgenesis

Transgenic animals raiseseveral particular moral issues

C Shouldthere be universal protocolsfortransgenesis?

C Is human welfaretheonly consideration? What aboutthe welfareofother lifeforms?

C Shouldscientistsfocuson in vitro (culturedin a lab) transgenic methods ratherthan,or

before, using live animalsto alleviate animal suffering?

C Will transgenic animals radically changethedirection ofevolution, which may resultin

drasticconsequencesfor nature and humans alike?

C Should patents be allowedon transgenic animals, which may hamperthefreeexchangeof

scientific research?

C Organ rejection!!

C Fail togiveobservablechange.

Religious viewsoftransgenic animals:

God laid down the structure of creation and any tampering with it is sinful


32/32

Interestingly, the creation of transgenic animals has resulted in a shift in theuse of laboratory animals, from the use of higher-order species such as dogsto lower-order species such as microbes, and has decreased the number of

animals used in such experimentation, especially in the development ofdisease models. This is certainly a good turn of events since transgenictechnology holds great potential in many fields, including agriculture, medicine,and industry.

CONCLUSION

THANK YOU !

knock out technology By Biksh

Documents

Transcript of knock out technology By Biksh