8/7/2019 Gene Knockdown
1/11
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 ???
8/7/2019 Gene Knockdown
2/11
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
8/7/2019 Gene Knockdown
3/11
8/7/2019 Gene Knockdown
4/11
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
8/7/2019 Gene Knockdown
5/11
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
8/7/2019 Gene Knockdown
6/11
8/7/2019 Gene Knockdown
7/11
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
8/7/2019 Gene Knockdown
8/11
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
8/7/2019 Gene Knockdown
9/11
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
8/7/2019 Gene Knockdown
10/11
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)
8/7/2019 Gene Knockdown
11/11
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.