gene knockdown

8/7/2019 Gene Knockdown

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Gene Knoc kdownMBB156: Ana lysis Session 8Ba y 4

Presented By Kiran Dedhia

Crea ted By Kiran Dedhia, Mkuzi,Nkhata, Charlotte Young , Ma xHeb ditc h, Rob yn Bradbury,Olympia Gianfranc esc o, Jam esCrowe and ???


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Gene knoc kd own is a tec hniq ue w hic h a llows theexp ression of one o r more g enes to b e red uc ed .

This c a n b e d one in tw o d ifferent w a ys. Genetic Mo d ific a tion.

This involves a p erma nent c ha nge in the DNA lea d ing top erma nently red uc ed gene exp ression.

Trea tment with a sp ec ific rea gent. A short p iec e of DNA or RNA w ith a seq uenc e c om p lem enta ry

to the gene or mRNA tra nsc rip t.

This lea d s to a temp ora ry red uc tion in exp ression Crea ting a tra nsient knoc kd own

Introduction


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Bind ing of oligonuc leotid ec a uses d ec rea d edexpression

DNA p rimer with

c om p lem enta ry seq uenc eto g ene b loc ks tra nsla tion Prevention o f tra nsla tion is

a c hieved b y RNA

interferenc e (RNAi) RNAi a c hieved through

miRNA a nd siRNA Co mp lem enta ry b ind ing

with the ta rget m RNAp rom otes d eg ra d a tion o fmRNA and b loc ks p re-mRNA splicing.

Transient Knoc kdown


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3 types of DNA b ind ings Minor g roove b ind ing (A)

Polyamid es b ind a t m inorg roove of DNA

Ma jor g roove b ind ing (C) Short DNA fra gment tha t

inte ra c t like Hoogsteen b a sep a irs a nd form trip le stra nd edDNA

PNA (pep tid e nuc leic a c id )b ind ing (B) Suga r-p hosp ha te ba c kb one

is rep la c ed b y 2-a minoethylglycine carbonyl

All p revent b ind ing of RNAp olymera se, tra nsc rip tionfac tors or induc e genesp ec ific d a ma ge to DNA

DNA b inding of oligonuc leotides


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Orga nisms in w hic h the exp ressionof a gene ha s b een red uc ed

Knoc kd ow n tec hniq ues a p p ea r to

b e ea sier to c a rry out tha nc om p lete knoc kout tec hniq ue

Orga nisms a re ma d e forinvestiga tion into the func tion of ap a rtic ula r gene, b y injec ting a nemb ryonic c ell w ith siRNAs whic hinterfere with tra nsc rip tion

Knoc kd own organisms


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INTRODUCTION

Reve rse gene tic s is the

op p osite of c la ssic a lgenetic s, or forwa rdgene tic s in tha t the g eneseq uenc e is id entified first

a nd a p henotyp e a ssoc ia tedwith this gene is the n workedout.

Sc ientists use reve rse gene tic sto c onnec t a g iven geneticseq uenc e with sp ec ificeffec ts on the orga nism.

DIRECTED DELETIONS AND

POINT MUTATIONS Site d irec ted mutagenisis is

used to c hange reg ula toryreg ions in a p romote r site or

ma ke sub tle c ha nges toc od on seq uenc es to the ORFto see whic h a mino a c idresid ues a re imp orta nt top rote in func tion.

This tec hniq ue c a n a lso b eused to m a ke g enenon func tiona l, the se a rec a lled null a lleles.

TECHNIQUES OF REVERSE GENETICS

Reverse Genetic s


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Gene silenc ing Using d oub le stra nd ed RNA a lso known

as RNA intereference (RNAi) Crea tes a sp ec ific knoc kout without a c tua lly muta ting the

DNA of interest. Ac ts b y d irec ting c ellula r systems to d eg ra d e mRNA

Morpholino Oligosaccharides Bind to b loc k ac c ess to ta rget m RNA w ithout req uiring the

a c tivity of c ellula r p rote ins

Effec tive in syste ms

Techniques


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Interferenc e using Tra nsgenes Molec ula r ge netic a p p roa c h

Orga nisms tha t over exp resses a norma l geneof inte rest OR

Orga nisms tha t over exp resses a muta nt form of a gene tha tinte rferes with w ild typ e func tion

Dominant nega tive intera c tion

Tec hniques (c ont)


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Future aspec ts

Post-transla tiona l silenc ing of g ene exp ressionis a p ow erful molec ula r tool for stem c ellresea rc h to stud y ge ne func tion,b iolog ic a l p a thwa ys a nd p hysiology of

d isea se. Gene silenc ing via siRNA ena b lesseveral applications:

Develop me ntal pa thwa y sc reening tod etermine the gene func tion a nd id entifytra nsc rip tion fac tors tha t regula te self-renewa l,

p rolifera tion, d ifferent ia tion a nd a p op tosis.

Direc ted d ifferent ia tion of emb ryonic or tissue-sp ec ific stem c ells b y silenc ing genes ofp lurip ote nc y ma intena nc e, suc h as

Oc t 4 or Na nog to induc e e a rly d ifferentia tionand linea ge d etermination.

Rep rogra m or d ed ifferentia te tissue sp ec ificstem c ells to repa ir d ama ged tissues or orga ns.

gene knockdown

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