2014 Predavanje Broj 9 Manipulisanje genima

8/11/2019 2014 Predavanje Broj 9 Manipulisanje genima

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Zakon o genetiki modifikovanimorganizmima

Genetiki modifikovan organizamje definisankao organizam ije su nasledne osobine

modifikovane intervencijom ovekaprimenombilo koje metode koja za rezultat ima uvoenjenovog, rearaniranje, rekombinaciju u genomu ili

eliminaciju genetikog materijala iz genomaorganizama, na nain koji se ne deava u prirodi.


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Transgeni organizmi

Organizmi kod kojih je vetakiuvedena DNK stabilno inkorporisana u

germinativne elije (gamete) ili uelije prekursore ijom deobom

nastaju gameti


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Genetiki modifikovani mikroorganizmi

A GM mikroorganizmi su virusi, bakterije ili kvasci ija jeDNA modifikovana na nain koji se ne deava u prirodi

Pogodni zbog relativno jednostavne strukture i male kompleksnostinaslednog materijala

Velika mogunost komercijalne primene i produkcije materija kojeprirodno ne proizvode

Pitanje primene

Pitanje potencijalnih opasnosti povezanih sa GM mikroorganizmima


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Primena GM mikroorganizama

Produkcija goriva i raznih industrijski i farmakoloki znaajnihjedinjanja (antibiotici, enzimi, dijagnostika jedinjenja)

Komercijalna proizvodnja insulina, interferona, hormona rasta,virusnih vakcina)

Biodegradacija i bioloko preiavanje (otpad, toksiniotpad...)

Dekontaminacija toksinih materija u zemlji ili vodi

Produkcija useva kroz bioloku kontrolu bolesti biljaka(virusne ili gljivine infekcije)


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Koje su potencijalne opasnosti vezane zaprimenu GM mikroorganizama?

Proizvodnja hrane zavisi od bakterija (sir, jogurt...) koje mogubiti modifikovane da se ubrza proces proizvodnje.

U veini sluajeva genetika modifikacija je dizajnirana dautie na proces prerade, ali ne i na sam produkt. Zbog stroge

kontrole i ograniene upotrebe, malo je verovatno da ovaprimena moe predstavljati opasnost za okolinu.

Za GM mikroorganizme razvijene u cilju zatite okoline postojibojazan od potencijalnog rizika za okolinu i zdravlje ljudi


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Genetiki modifikovane bakterije

Prva primena ostvarena 1978.g.: Herbert Boyer (University of California) ubaciohumani gen za insulin u bakteriju Escherichia coliipoeo proizvodnju sintetikog "humanog" insulina

(Humulin).

Insulin

Vakcina za Hepatitis B

Plasminogen aktivator

Humani faktor rasta1982. g. Humulin,

postaje prvirekombinantni DNK

protein prihvaen odstrane FDA


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Ekspresija humanoginsulina u E.coli

Dva lanca insulina seeskprimiraju odvojeno kao

fuzioni proteini sa -galaktozidazom

Dobijeni proteini se procesujuhemijskim putem i meaju ucilju dobijana aktivne forme

proteina


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Genetically modifification of plants

Insertion of isolated individual gene(s) into the genomeof the plant, usually using plant tissue that has been

prepared to take up the gene(s)

Regenerating an intact plant from the geneticallymodified plant tissue

The gene should be inherited by the offspring of thenext generaton

If the introduced genes are functional (inserted genes

should work as expected) and the gene-productsynthesized, the plant is said to be transformed.


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How are foreign genes insertedinto plants?

The Ti Plasmid and Agrobacteriumtumefaciens

ProtoplastsThe Gene Gun


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Agrobacterium tumefaciens

Gram-negative soil bacterium, a naturally occurring plant pathogenicbacteria.

As a normal part of its life cycle genetically transform plants

Causes crown gall disease of a wide range of dicotyledonous plants

The disease gains its name from the large tumour-like swellings(galls) that typically occur at the crown of the plant, just above soil

level

Basically, the bacterium transfers part of its DNA to the plant, andthis DNA integrates into the plants genome, causing the production

of tumours and associated changes in plant metabolism.

Most of the genes involved in crown gall disease are not borne onthe chromosome of A. tumefaciensbut on a large plasmid, termed

the Ti(tumour-inducing) plasmid.


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Ti plasmid of Agrobacterium tumefaciens

A large circular DNA plasmid (200 kb) Has the ability to enter plant cells and insert

a portion of its genome into plantchromosomes: when the bacterium infects aplant cell, a part of Ti plasmid called T-DNAis transferred and inserted, more and less, atrandom into genome of host plant

T- DNA is transferred as a linear, singlestranded molecule and eventually becomesintegrated in the plant chromosomal DNA

The insertion of the Ti DNA into plant

genome depends on specific sequence at theright border Virgenesare essential for the transfer and

integration of T-DNA region (7-8 genes) Most of the genes that are located within T-

DNA region are activated only after T-DNAis inserted into plant genome

Opinesare synthesized by the host plantunder the direction of the T-DNA. Thebacterium then uses the opines for its ownpurpose

Both auxin and cytokininregulate plantgrowth and development and in excess cancause the plant to develope tumour growth


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Ti plasmid derived cloning vectorsystems

The Ti plasmid has been engineered to make it a vectorfor plant transformation by including sequences for

replication in E. coliand Agrobacterium, uniquerestriction sites for inserting foreign genes, and

selectable markers.

The binary cloning vector

The cointegrate cloning vector


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The binary cloning vector


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Cointegrate vector - carries onlyE.coliorigin of replication andcan not exist autonomouslywithin A. Tumefaciens. Containsselectable markers, T-DNA right

border, a target gene andsequence that is homologous to asegment on the disarmed Tiplasmid

Disarmed Ti plasmid-contains T-DNA left border, the virgenecluster, and A. Tumefaciens ori.

The final recombinant plasmidhas T-DNA left and right

borders bracketing the clonedand plant reporter genes


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Production oftransgenic

plantsIntermediate

vector

Disarmed Ti plasmid

Cointegrate

plasmid


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Protoplasts

Protoplasts are cells that have had their cell walls removed. This can

be done mechanically, or by enzymic digestion. The 'naked' cells aresurrounded only by a cell membrane and can be used in a variety of

ways. Two or more protoplasts can be fused with the help of a detergent, polyethylene

glycol, to produce hybrid cells with characteristics from each 'parent'.

Infection of protoplasts with genetically-modified Agrobacterium tumefaciensis

one way of introducing new genes into plant cells. Whole plants can be regenerated from protoplasts grown on solid or liquid media.

PEG (PolyEthylene Glycol): PEG mediated DNA uptake- polyethylene glycolpermeabilizes protoplast membrane allowing DNA to pass through.

Electroporation: a brief, high voltage direct current pulse applied to protoplastsmakes membrane permeable to high molecular weight substances, such as DNA,

RNA and viruses. Microinjection: micro-needles are used to inject DNA directly into nucleus


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Gene-gun The gene gun method can be used

with all plant species.

This uses gold or tungsten (volfram)microparticles, coated with

transgene DNA, which are fired intothe target tissue by an explosivedischarge or pressurized helium.

DNA that penetrates the nucleus ofthe plant cell may be incorporated

among the plant's own genes.

The gene may be expressed but

often only temporallly, very often itis not truly incorporated into thegenome and will not be expressed in

the following generation.


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Transgenic strategies being exploredor commercialised

The transgenic tomatoes do not express the gene for polygalacturonase, anenzyme that degrades pectin, leading to softening of the fruit tissues

Several crop plants have been engineered to express the insecticidal toxin geneof Bacillus thuringiensisso that insects attempting to eat these plants are

killed (potential disadvantage -selection for the development of toxinresistance)

Several crops also have been engineered for resistance to herbicidessuch asglyphosate, so that the herbicide can be used for weed controlwithoutdamaging the crop ("Roundup Ready" crop plants marketed by Monsanto)

Engineering for virus resistanceby incorporation of viral coat protein genes orantisense RNA

Engineering for resistance to fungal pathogens, by enhanced expression offungal wall-degrading enzymes (chitinase and glucanases)

Engineering of plants so that, during a late stage in the development of theirseeds, they express a gene that makes the seeds sterile("terminator

technology)

S h p lj nj n tiki


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Svrha pravljenja genetikimodifikivanih biljaka

Poveanje prinosa

Poboljanje hranljive vrednosti

Biljke kao ivi bioreaktori za jeftinu proizvodnju vanihproteina i metabolita

Stvaranje biljaka sa novom, poeljnom kombinacijomosobina:

Otpornost na insekte

Otpornost na viruse

Otpornost na herbicide

Odloeno cvetanje

Otpornost na suu i stres (visok salinitet, temperaturni ekstremi,hipoksija, nedostatak minerala, toksini metali, poveana UV radijacija)


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Biljke rezistentne na insekte

Ako biljke produkuju insekticide postae otporne nainsekte i nee biti potrebe za zapraivanjem skupim i

tetnim insekticidima

Prirodni insekticidi su visoko specifini, ogranieni na

mali broj vrsta i nisu tetni za ljude Glavne strategije:

Uvoenje gena za insekticidni PROTOXIN iz Bacillus thuringiensis

Korienje gena za inhibitore proteaza. Njihovo prisusvo ograniavamogunost insekata da svare hranu, pa je konzumiranje hrane umanjeno


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Biljke rezistentne na viruse

Virusi oteuju biljke i znaajno umanjuju prinos

Strategije:

1. Transfer prirodnih gena koji dovode do rezistencije na

viruse iz jedne vrste u drugu Dolazi do spontane reverzije Rezistencije su visoko specifine

2. Vakcinacija biljaka genima za omotoe virusa, drugimviralnim genima ili korienjem antisens sekvenci za

gene virusa


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Biljke rezistentne na herbicide

Oko 10% biljne produkcije u svetu se gubi zbog pojavekorova i godinje se troi oko 10 milijardi dolara za oko

stotinu vrsta razliitih hemijskih herbicida Stretegije:

1. Spreiti unos herbicida2. Hiperprodukcija target proteina koji je osetljiv na

prisustvo herbicida da bi ostala dovoljna koliina tihproteina neophodnih za odravanje normalnih funkcija u

eliji

3. Ometanje vezivanja target proteina osetljivog naprisustvo herbicida za sam herbicid4. Razvoj biljaka sa mogunou inaktivacije herbicida


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Kontrola razvojnih i fiziolokih procesa

cvetanje

vegetativno razvie i arhitektura

odgovor na hormone

opadanje i suenje lia

self-inkompatibilnost

fototropizam........

bre do ploda

lepota u razliitosti

GM biljke kao bioreaktori


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GM biljke kao bioreaktoriBiofarmaceutski proizvodihormon rasta

eritropoetininterferonhumani serum albumin-1-antitripsinhirudin

glukocerebrozidaza

vakcine i monoklonska antitela

industrijski enzimilaktoferin, kazein, amilaza, fitaza, hidrolaza

biodegradabilna plastika i proteinski polimeriElastin, kolagen

eksperimentalni model-duvan


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ZLATNI PIRINAsadri -karotenoptimalna doza gvoa

uti narcis

Erwinia

4 gena za sintezu

-karotena2

2 X

gljiva

bob

pirina

fitaza

feritin

metalotionein


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VAKCINE ANTITELA PLANTIBODIES

jestive vakcine

kliniki testirane:

-hepatitis B-dijarea uzrokovanaenterotoksinom iz

E.colii virusomNorwalk-ap i slinavka goveda-gastroenteritis svinja

epitop specifine dijagnostikaterapija zaraznih bolesti ikancera

kliniki testirano:-IgA antitela protivStreptococcusMutans izazivaa karijesa

(duvan)-antitela zaimunodijagnostikukancera

GM biljk i d lj


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GM biljke i zdravlje

? Da li ugraeni gen kodira protein koji moe bititoksian ili izazivati alergije

? Da li je ugraivanje dovelo do neeljenog

poveanja ekspresije gena biljke domainaiji proizvod ima toksino dejstvo

? Da li ugraeni gen moe izazvati promenu

mikroorganizama koji naseljavaju humani digestivnitrakt i tako posredno ugroziti oveka

GM biljke i ivotna sredina


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GM biljke i ivotna sredina

Da li moe doi do transfera gena kojim je

izvrena modifikacija biljkena druge organizmetransfer na druge biljketransfer na mikroorganizme

?

Kakav je efekat proteina kodiranogtransgenom na ne-ciljne organizme

?

Da li GM biljke imaju selektivnu prednost kojom ugroavajudruge biljne vrste i da li vre selektivni pritisak napopulacije mikroorganizama, insekata i ostalih tetoina?

?


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Genetikimodifikovane biljke - za i protiv

GMO

POLITIKAETIKAI RELIGIJA

BIOLOGIJAEKONOMIJA


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Transgenic animals

A transgenic animal is one that carries a foreign genethat has been deliberately inserted into its genome.

In addition to a structural gene, the DNA usuallyincludes 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

Three methods of producing transgenic mice arewidely used:

Retroviral vector method

The Pronucleus Method

The Embryonic Stem Cell Method



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An effective means of

integrating the transgene intothe genome of recipient strain

Can only transfer small pieceof DNA (up to 8 kb) and may

lack adjacent regulatorysequences

Retroviruses integrate atrandom

Rarely used for creatingtransgenic animals that havecommercial use



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Females aresuperovulated andmated with malesOocytes are recoveredfrom excised oviductsDNA is microinjectedinto male pronucleus

Surviving oocytes arereimplanted into theoviducts of fostermother

The Embryonic Stem Cell Method


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Transgenic mice can be

established by crosses

from founder mice that

carry transgene in their

germ lines


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Gene targeting

Engineering mutation in a preselected gene within anintact cell

It is a form of artificial site-directed in vivo

mutagenesis The mutation may result in inactivation of geneexpression (knock-out) or altered expression and is used

for studying gene function

Gene targeting typically involves introducing mutation by

homologous recombination


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Homologous recombination

Nonspecific integration

US: unique segment

TG: transgene

HB: homologous blocksCS: complementarychromosomal sites

Targeted mutation: gene knock-out


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arg t mutat on g n noc out

The gene is inactivated byinsertion of neo geneinto

protein coding region

The vector has the secondmarker- herpes tk gene

To isolate cells carrying atargeted mutation all cells

are put into mediumcontaining neomycin analog(G418) and ganciclovir

G418 eliminates cells inwhich no integration ofvector has occurred

Ganciclovir kills cells thatharbor the tk gene therabyeliminating cells bearing arandomly integrated vector

Inactivation of gene in desired cell type: cre-


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g yploxP recombination system

CRE: causesrecombination

Mediate recombinationbetween two loxPsequences that are in thesame orientation leading toexcision of the interveningsequence

M: markerA: target locus

Transgenic domestic


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Transgenic domesticanimals

GloFish: the first genetically modified


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GloFish: the first genetically modifiedanimal to be sold as a pet
http://upload.wikimedia.org/wikipedia/commons/f/f2/GloFish.jpg

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2014 Predavanje Broj 9 Manipulisanje genima