Docsity
Docsity
Prepare for your exams
Prepare for your exams

Study with the several resources on Docsity

Earn points to download
Earn points to download

Earn points by helping other students or get them with a premium plan

Guidelines and tips
Guidelines and tips
Sell on Docsity
Sell on Docsity
Docsity AI
Docsity AI
Log inSign up

Prepare for your exams

Study with the several resources on Docsity

Find documents

Prepare for your exams with the study notes shared by other students like you on Docsity

Search for your university

Find the specific documents for your university's exams

Docsity AINEW

Summarize your documents, ask them questions, convert them into quizzes and concept maps

Explore questions

Clear up your doubts by reading the answers to questions asked by your fellow students

Earn points to download

Earn points by helping other students or get them with a premium plan

Share documents
download point icon20 Points

For each uploaded document

Answer questions
download point icon5 Points

For each given answer (max 1 per day)

All the ways to get free points
Get points immediately

Choose a premium plan with all the points you need

Study Opportunities

Choose your next study program

Get in touch with the best universities in the world. Search through thousands of universities and official partners

Community

Ask the community

Ask the community for help and clear up your study doubts

Free resources

Our save-the-student-ebooks!

Download our free guides on studying techniques, anxiety management strategies, and thesis advice from Docsity tutors

Gene Function Testing: Guide to Targeted Knockouts & RNA Interference, Study notes of Biochemistry

University of Wisconsin (UW) - MadisonBiochemistry

An in-depth exploration of gene function testing, focusing on targeted gene knockouts using homologous recombination and rna interference. Various model systems, assays, and techniques for testing gene function, as well as generating knockout mice and characterizing knockout phenotypes. Additionally, it discusses other gene knockout approaches and rnai mechanisms.

Typology: Study notes

Pre 2010

Uploaded on 09/02/2009

koofers-user-jcz
koofers-user-jcz 🇺🇸

10 documents

1 / 12

Toggle sidebar

This page cannot be seen from the preview

Don't miss anything!

bg1
Testing gene function
Biochemistry 660
Bryan Phillips
September 17th, 2008
[email protected]
Introduction
Targeted gene knockouts
RNA interference
Outline
Biology in the post-genomic era
~25,000 human genes
– Many of unknown function
– 21st century gold rush:
“So many genes, so little time”
Model systems for studying gene function
Mammalian cells
E. coli S. cerevesiae
Mice
Xenopus
Drosophilia
C. elegans
Zebrafish
Arabidopsis
Ciona intestinalis
pf3
pf4
pf5
pf8
pf9
pfa

Partial preview of the text

Download Gene Function Testing: Guide to Targeted Knockouts & RNA Interference and more Study notes Biochemistry in PDF only on Docsity!

Testing gene function

Bryan Phillips^ Biochemistry 660

[email protected]^ September 17th, 2008

RNA interferenceTargeted gene knockouts Introduction

Outline

Biology in the post-genomic era

~25,000 human genes

Many of unknown function

21st century gold rush:

“So many genes, so little time”

Model systems for studying gene function

E. coli Mammalian cells

S. cerevesiae

Mice

Xenopus Drosophilia

C. elegans

Zebrafish

Arabidopsis

Ciona intestinalis

  • Assays: ?

?

?

Testing gene function

YFG

(Your Favorite Gene)

Testing gene function

YFG

expressed YFG

Assays:

•Antibody stain•Western blot•RT-PCR•In situ hybridization•Northern blot

Testing gene function

YFG mutant

Wild-type

Assays:

Testing gene function

Misexpress YFG

Wild-type

Role in cell cycle progression?

Assays:

Gene knockout by homologous recombination

regions flanking YFGIntroduce a selectable marker flanked by sequence homologous to

YFG replaced by selectable marker

YFG (Your Favorite Gene) Selectable marker Selectable marker

Homologous recombination occurs

KanMX General scheme for HR in yeast

KanMX

KanMXYFG KanMX

Confirm knockout by PCRcolonies.Use selection to isolate knockoutproductTransform yeast with the PCRYFG homology PCR amplify marker to contain

Generating knockout mice

modifications in mice by the use of embryonic stem cells” “for their discoveries of principles for introducing specific gene

Medicine 2007The Nobel Prize in Physiology or

Mario R. Capecchi

Sir Martin J. Evans

Oliver Smithies

Generating knockout mice

marker.replace YFG with selectableUse homologous recombination to

event.Select for the recombination

early mouse embryoInject transformed ES cells into

YFG

Neomycin

r YFG(+)Embryo cells

YFG(-)ES cells

Early mouse embryo

addedwith NeomycinCell culture

“Chimeric” mouse embryo

Implant the recombinant embryos

mate with wild-type miceIdentify YFG(–) chimeric mice and

each otherIdentify YFG(-) carriers and cross to

Identify YFG(-) homozygotes

Generating knockout mice

YFG(+) Embryo cells

YFG(-)ES cells

Early mouse embryo

x wild-type mouse: YFG(+)

+/+ YFG(+) or YFG(-)chimeric mouse:

+/-

+/-

+/+

+/+

+/-

+/-

-/-

Implant the recombinant embryos

mate with wild-type miceIdentify YFG(–) chimeric mice and

each otherIdentify YFG(–) carriers and cross to

Identify YFG(–) homozygotes

Generating knockout mice

YFG(+) Embryo cells

YFG(-)ES cells

Early mouse embryo

x wild-type mouse: YFG(+)

+/+ YFG(+) or YFG(-)chimeric mouse:

+/-

+/-

+/+

+/+

+/-

+/-

-/-

Coat cells

Germline cells

Characterizing your knockout

Characterizing knockout phenotype

  • Possible issues: ?

Confirm knockout

  • Protein levels– mRNA levels

Confirm specificity

Neighboring genes

Rescue of knockout phenotype

“Flox” region of interest

promoter)(i.e. from tissue-specificmediated recombinationCre recombinase-

DNA between

(^) loxP

(^) sites

is excised

Conditional knockouts

loxP

loxP

Gene knockouts: system comparison

Days

Fastest

E. coli

1-2 years

Slowest

Mice

Months

Slower

Arabidopsis

Weeks

Fast

C. elegans

Days

Faster

Yeast

Time

Speed

Organism

Ordering gene knockouts

  • Mouse: (^) International Mouse Strain Resource

www.informatics.jax.org/imsr/index.jsp

Jackson Labs

C. elegans:

(^) Gene Knockout Consortium

www.celeganskoconsortium.omrf.org/www.grs.nig.ac.jp/c.elegans/index.jsp

Yeast:

(^) Saccharomyces

(^) Genome Deletion Project

www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html

  • Drosophila melanogaster (^) Resources listed in

(^) Nature Reviews Genetics

(^) 2005,

(^) 6 : 179-

  • Arabidopsis thaliana http://www.arabidopsis.org/portals/mutants/worldwide.jsp^ T-DNA insertion and knockout resources listed at:

RNA interference: Target is YFG mRNA Targeted gene knockouts Introduction

–Harnessing–vs. miRNA–Mechanism

Outline

Medicine 2006The Nobel Prize in Physiology or

double-stranded RNA”“for their discovery of RNA interference- gene silencing by

Mechanism of RNAi: Step 1

dsRNA

5’P, 3’ 2-nt overhangssiRNAs: 19-25 nt long:

Meister & Tuschl,

(^) Nature (^) 431 : 343-

SourcedsRNA

P
P
DCR-
ATP

Mechanism of RNAi: Step 2

Meister & Tuschl, siRNA-Dicer complex

(^) Nature (^) 431 : 343-

P
P
DCR-
P

Ago

ATP

Ago

P
RISC

Mechanism of RNAi: Step 3

Meister & Tuschl,

(^) Nature (^) 431

: 343-349^ YFG mRNA

P

Ago

P

Ago

AAAA

7mG

mRNA cleavage

Drosha

pre-miRNA

nucleus

small RNAsEndogenousmicroRNAs:

exportin-

cytoplasm

5’ cap

AAAA

pri-miRNA

Meister & Tuschl,

(^) Nature (^) 431 : 343-

Thermodynamic considerations:siRNA design guidelines (mammalian cells)

passenger strand^ guide strand

5’ end unwinding

into RISCguide strand incorporated

  • Overall low to medium G+C content (30-52%)

Base preferences: -Presence of an A or U at position 10 of guide strand- C or G at position 19 of guide strand- U or A at position 1 of guide strand- Guide strand: low internal stability (GC content) at 5’ end

high internal stability (GC content) at 3’ end

dsRNA^ Mechanism of RNAi: Step 1

5’P, 3’ 2-nt overhangssiRNAs: 19-25 nt long:

Meister & Tuschl,

(^) Nature (^) 431 : 343-

P
P
DCR-

Source dsRNA

ATP

Approaches for RNAi: #1. Synthesized siRNAs

Order siRNAs

Transfect siRNA into cells

UU

UU

UU

UU UU

UU

UU

UU

UU

UU

YFG mRNA

P

Ago

AAAA

7mG

YFG mRNA cleavage

RISC

(shRNA) expression vector Approaches for RNAi: #2. Small hairpin RNA

P

P

DCR-

Puromycin

(^) r

pol III promoter

YFG mRNA TTTTTTT

P

Ago

AAAA

7mG

YFG mRNA cleavage

ATP

DCR

RISC

Comparison of RNAi approaches

Transfected siRNAs

shRNA vectors

Disadvantages: Advantages:

  • Must optimize conditions- Expensive- Transient - Faster- Simpler approach

(unless ordered)- Require cloning- Can be inducible- Stable(if homemade)- Less expensive

Confirming and optimizing RNAi

  • Controlling for off-target effects • Validate RNAi knockdown
  1. Homology to other genes

a) Rescue

  • RT-PCR or Northern blot- Western blot

Confirming and optimizing RNAi

  • Controlling for off-target effects • Validate RNAi knockdown
  1. Homology to other genes

b) Multiple siRNAsa) Rescue

  • RT-PCR or Northern blot- Western blot

Confirming and optimizing RNAi

  • Controlling for off-target effects • Validate RNAi knockdown

a) Minimal transfection dose

  1. Innate immune responses (e.g. Interferon response)2) Saturating RNAi pathway1) Homology to other genes

b) Assay pathway protein/mRNA levelsa) Control siRNAb) Control siRNAb) Multiple siRNAsa) Rescue

  • RT-PCR or Northern blot- Western blot