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
Retroviral vector method
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Retroviral vector method
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
The Pronucleus Method
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The Pronucleus Method
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