Expression and Function

8/13/2019 Expression and Function

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Studying gene expression and

function


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Mutagenesis and protein engineering

Possible to generate mutants anddetermine the phenotypes of resultantproteins

Can be done both in vitro and in vivoEthylnitrourea (ENU) is used to generate

mutations in vivo (mainly mice)

The mutations generated are randomIn vitro mutagenesis enables contol of the

products generated


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Recombination

Three main types

Homologous recombination takes place between

2 highly identical sequences

Site specific recombination: between sequencesthat have short stretches of homology (invertible

elements, resolvases, phage DNA intergration)

Non-homologous (illegitimate) recombination:sequences with no homolgy at all (transiposition,

xsome deletions, inversions, translocations)


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Mechanism of homologous recombination

http://www.blackwellpublishing.com/trun/artwork/Animations/Recombination/recombination.html

Strands align

Nicks in the DNAs

Strands displace to opposite

DNA

Ligation covalently links theDNAs

(Holliday structure)

The branch point migrates

DNA cleaved again, new DNAmolecules formed


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Homologous recombination and gene

knock-outs

Can be used to generate a gene knock-out

The method of choice for replacing one allele

with an engineered construct without affecting

any other locus in the genome .The DNA sequence of the gene to be replaced

as well as adjoining region has to be known.


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knock-outs

Plan to Insert different alleles (both functional and non-functional

ones),

Different genes or reporter genes (e.g. antibiotic resistanceor green fluorescent protein).

Include some flanking DNA that is identical in sequence tothe targeted locus.

A negative selection marker (e.g. thymidine kinase, tk)may be added to the replacement vector.

The negative marker is outside the region of sequencesimilarity between the vector and the targeted locus.


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knock-outs

Then introduce construct into the cell Heat shock

Electroporation

Gene gun

Agrobacterium

Plant virus

During cell division

Construct aligns along the targeted gene

Recombination takes place within the homologoussequences

The recombination may take place anywhere within theflanking DNA sequences

The exact location is determined by events in the cells.


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Homologous recombination andgene knock-outs

In this case the negative marker does not get

integrated


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Cells that incorporated the construct are selected byrespective antibiotic in medium (positive selection)

Non-homologous recombination offspring are eliminatedby addition of antibiotic as well as gancyclovir in medium

Thymidine kinase activates Gancyclovir, a nucleotideanalog, by addition of phosphate.

Activated Gancyclovir gets incorporated, the cells die


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knock-outs

Sucess of gene knock is affected by ploidyFor haploid organisms, only one round of

tranformation is necessary

For diploids, 2 steps involving 2 differentselection markers are desired

Knock-outs in polyploids are not practical

(need to use several antibiotics!!)Its difficult to target Multiple copy genes


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Plan for gene knock-out from

diploid cell in vitro

Make 2 constructs each with a different marker

Transform with construct carrying first resistance

marker

Select with the respective antibiotic

Transform resultant heterozygotes with second

construct carrying a different marker

Select with both antibiotics (use antibiotics withno known cross resistance)

Confirm correct intergration by southern blots


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Gene knock-out in mice

Homologous recombination to replace one

allele

Followed by two or more generations of

selective breeding until a breeding pair are

isolated that have both alleles of the

targeted gene inactivated (knocked-out).

The role of a particular gene is determined

by observing the knock-out phenotype.


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2 approaches

Can transform embryonic stem cells

Inject construct into newly fertilised egg


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Isolate developing

embryo at blastocyst

stage. This embryo is

from a strain of micewith gray fur.

Remove embryonic

stem cells from gray-

fur blastocyst. Growstem cells in tissue

culture.


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Transfect stem cells

with construct.

Select for

homologousrecombination by

growing stem cells in

neomycin and

gancyclovir.+ neomycin + gancyclovir


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Remove homologously

recombined stem cells

from petri dish and inject

into a new blastocyst thatwould have only white fur.

Implant several chimeric

blastocysts into pseudo-

pregnant, white furmouse.


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Mother will give birth to a

range of mice.

Normal white fur and

Chimeric mice with some oftheir cells derived from

recombinant stem cells.

Fur cells from recombinant

stem cells produce gray

patches.


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Mate the chimeric micewith wild-type white furmice.

If the gonads of thechimeric mice were derivedfrom recombinant stemcells,

all the offspring will havegray fur.

Every cell in gray mice areheterozygous for the

homologous recombination.


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Mate heterozygous gray

mice (+/ H) and genotpye

the gray offspring.

Identify homozygousrecombinants (H / H) and

breed them to produce a

strain of mice with both

alleles knocked out.

The pure breeding mouse

strain is a "knockout

mouse". .


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Knock-outs in plants

Discs cut from a leaf

Incubated with engineered

Agrobacteria for 24 hrs

Selection for cells withintergrated constructs

Addition of shoot inducing

medium

Transfer to root inducingmedium

Grow seedling into adult plant

with transgene.


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Antisence DNA oligonucleotide

technology

Injecting a cell with antisense DNA oligonucleotidesRNA-DNA hybrids form

Degradation of hybrid by RNAse H

Abolished expression of the respective protein by

depleting the cytoplasm of mRNA


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The antisense RNA approach

Similar effect can be

achieved by engineering the

cell to generate dominant

negative mutations

Can be done by cloningreverse complement of part

of the gene in tandem

ATGCTTTGCAT

Resultant RNA folds ontoself to make double strand

This is the basis for RNA

interference (RNAi)


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RNA interference

A naturally occuring post-transcriptional

gene silencing phenomenon

Evolutionariry conserved

Sequence specific

We can use RNAi to answer the question:

Is the gene essential?


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RNA interference

RNAi is also known as knock-down

Applications includeIn vitro and in vivo gene targeting

for functional studies of genes known to be

up-regulatedFor identification of drug targets, leading to

novel therapeutic strategies


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RNAi has potential in medicine

Cancers are charaterised by upregulation

of groth factors and growth factor

receptors

Novel therapeutic interventions would

target inhibition of such proteins or their

down regulation

f f ( )


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Mechanism of RNA interference (RNAi) in

mammalian systems.

M h i f RNA i t f (RNAi) i


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Mechanism of RNA interference (RNAi) in

mammalian systems.

Long double-stranded RNA molecules (dsRNA), which are expressed from DNA vectors (left red arrow) or directly enter the cell (center

red arrow), are processed by the Dicer complex resulting in the

formation of small inhibitory RNAs (siRNAs). Alternatively, to induce RNAi

these small 2123 bp duplexes are directly delivered into the

cell (right red arrow). The siRNAs are incorporated into a nuclease-containing multiprotein complex called RISC, which becomes activated

upon the ATP-dependent unwinding of the siRNA duplex by an RNA

helicase. The now single-stranded siRNA guides the RISC complex to

its complementary target mRNA which is then cleaved by the

endonucleolytical activity of RISC. While the RISC complex is recovered for

further cycles, the cleaved mRNA molecule is rapidly degraded due to its

unprotected RNA ends.


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Small interfering RNAs versus small temporal RNAs Double-stranded siRNAs of

length 2123 nucleotides are produced by Dicer from dsRNA silencing triggers.

Characteristic of RNase III products, these have two-nucleotide 3' overhangs

and 5'-phosphorylated termini. To trigger target degradation with maximum

efficiency, siRNAs must have perfect complementarity to their mRNA target (with

the exception of the two terminal nucleotides, which contribute only marginally torecognition). stRNAs, such as lin-4and let-7, are transcribed from the genome

as hairpin precursors. These are also processed by Dicer, but in this case, only

one strand accumulates. Notably, neither lin-4nor let-7show perfect

complementarity to their targets. In addition, stRNAs regulate targets at the level

of translation rather than RNA degradation. It remains unclear whether the

difference in regulatory mode results from a difference in substrate recognition orfrom incorporation of siRNAs and stRNAs into distinct regulatory complexes.


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The degradation of endogenous mRNA in a sequence-specific manner can be

induced by dsRNA [RNA interfernce (RNAi)], antisense transcription, or viral

infection. In the current model for posttranscriptional gene silencing by RNAi,

the ribonuclease III like enzyme Dicer processes dsRNA into small fragments

(siRNA) of 21-25 nts. The siRNA is incorporated into the RNA-induced silencing

complex (RISC), which targets and degrades the homologous mRNA. Most of

the proteins in the RISC (with the exception of Ago2 and Dicer have not been

identified) but likely contain endonuclease, helicase, exonuclease and

homology scanning activity.


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Delivery systems for RNAi

Constructs incorporated into

Liposomes: Lipid micelles that fuse with cell membranes

contents get delivered to the cytosol

Polyethylenimine

Then injected in mammal

Intraperitoneally,

Intravenous

Intrathecal

Intratumoral, e. t. c


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Polyethylenimine (PEI)-mediated

siRNA transfer


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T. brucei as a model for RNAi

MARKER

AAAA

MARKER

Inducible stem-loop or

opposing T7 promoters

DsRNA processed by ds

endonuclease to 21ntfragments with short

overhangs)

Ds fragments in RISC

complex; target RNA by

base-pairing

Target RNA destroyed


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The first RNAi in T. brucei1998

Double-stranded RNA with alpha tubulin

sequences in trypanosomes

Cells get round and stop growing


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Advantages of RNAi

Quick

Simple

Can target multi-copy genes even if

they are spread around the genome


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Disadvantages of RNAi

The RNA and protein are not eliminated Very often 10-15% of the normal level is

sufficient. If that is how the RNAi works, no

change in phenotype will be seen.

Leakiness - control by tetracycline

may be incomplete

Often there is some dsRNA made even in

the absence of tetracycline. If a small reduction in the protein has

deleterious effects, no live cells will be

obtained at all.


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Disadvantages of RNAi

Escapersfrom deleterious RNAi

After tetracycline is added, the cells initially show

effects but after about 5 days, they recover and look

normal

During the initial selection, cell lines are obtained but

the RNAi mysteriously doesntwork.

The cell lines look great for a couple of weeks of

culture but after that the RNAi doesntseem to work

any more

The cell lines look great and are frozen down. When

you thaw them out the RNAi no longer works.

Expression and Function

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