1

Day5

MOD1–DNAENGINEERING

Engelward,Fall2009

Lecture 1: Intro to importance of HR Polymerases & PCR

Lecture 2: How HR works Overview of experiments & discussion of controls (single digests)

Lecture 3: Why understanding matters: HR & BRCA2 Overview; Running an agarose gel; Purification from a gel; Discussion of controls

Lecture 4: Exploiting Scientific Understanding for Engineering: Achilles heel/Parp Inhibitors & Drug delivery Ligase and transformation & Data discussion (purified fragments)

Lecture 5: DNA Engineering in Mammals: Gene Targeting & Transgenics (Sonoda prep) Overview & Strategies for DNA analysis

Lecture 6: DNA Engineering: Conditional Expression (Sonoda Prep)

Lecture 7: DNA Engineering Reveals HR Function: Discussion of Sonoda

Lecture 8: Flow Cytometry: How it works and how to do it

GoingfromUnderstandingtoSolu6ons

‐Exploi6ngUnderstandingofHRforgene6cengineering

Mod1Overview:MethodsandLogic

‐Logicforstepssofar‐Strategyforanalysis

Why you owe Your Youthfulness to Homologous Recombina8on… 

LossofHelicase→FaultyRecomb.

Werner’sSyndrome

2

Why you owe Your Life to Homologous Recombina8on… 

TurnOffHomologousRecombinaLon→ChromosomesFallApart

Sonadaet al., EMBO J.17,598–608(1998).

Normal Rad51‐/‐

Why you owe Your Health to Homologous Recombina8on… 

DefecLveHomologousRecombinaLon→Cancer

Why you owe be;er  Cancer Treatments to HR… 

NewdrugtargetsPARPtokillBRCA2‐/‐tumorcells

NanocellforDrugDelivery

3

Why you owe Your Life to Homologous Recombina8on… 

But how do we that cells  cannot survive without HR? 

Sonadaet al., EMBO J.17,598–608(1998).

Normal Rad51‐/‐

TounderstandhowtheexperimentsweredonetoshowthatHRisessen6al,youneedtounderstand:

a)GeneTarge6ngb)Condi6onalExpressionc)CellCycleAnalysisbyFlowCytometry

GeneTarge6ng

GeneTarge6ngisallaboutexchangingDNA…

Buthowdoweswaponepieceforanother?

SeeSDSAAnima6onbyJus6nLo

4

NHEJ HR

DoubleStrandBreaks

KUheterodimer(Ku70&80)DNA‐PKcsDNAligaseIVXRCC4

E. coli:RecA*

Yeast:Rad51

Mammals:RAD51

Egelman, PNAS 2003

5

See Prototypic Model Animation by Justin Lo

6

HomologyDirectedRepair(HDR)Canleadtoexchangeofflankingsequences

Thisisthe‘prototypic’modelofhowhomologous

recombinaLoncanrepairadoublestrandbreak

Howcanweexploitourunderstandingof

HR&NHEJforGene6cEngineering?

Gene6cEngineeringinMice:

1) GeneTargeLng‐Turninggenesonandoff

2)Transgenics

‐InserLnggenes

Traditional Knock-Out Technology

Targeted Homologous Recombination

Your Favorite Gene

Neo hsvTK Long Arm Short Arm

Your Favorite Gene Neo

7

Neo hsvTK Long Arm Short Arm

Neo hsvTK Long Arm Short Arm

Traditional Knock-Out Technology

Random Integration

8

Traditional ES Knock-Out Technology

Engineered cells mix with recipient blastocyst to make a chimeric mouse

Chimeras

Inner Cell Mass

Implant into surrogate Inject ES cells from selected

clones into blastocysts

Day 2 Blastocyst

ES Cells

Needle

Characterize Clones Inject Three Clones

Drug Resistant Colonies

G418 FIAU

Targeting Vector

Electroporate

Chimeras

Inner Cell Mass

Implant into surrogate Inject ES cells from selected

clones into blastocysts

Day 2 Blastocyst

ES Cells

Needle

Characterize Clones Karyotype Inject Three Clones

Drug Resistant Colonies

G418 FIAU

Targeting Vector

Electroporate

Methods&LogicForMod1

9

X E PCR 1

E X

Purif. X E 2

E X

Digest X E 2

E X

Gel Purif. X E

3

Gel Anal.

3

Plan Lig.

3 X E

Ligate 4

E. coli E. coli

E. coli E. coli

Transform 4

X E PCR 1

E X

Purif. X E 2

E X

Digest X E 2

E X

Gel Purif. X E

3

Gel Anal.

3

Plan Lig.

3 X E

Ligate 4

E. coli E. coli

E. coli E. coli

Transform 4

Purif. DNA

5

Digest 5

Gel Anal.

6 Transfect

7

Flow

8

Howcanyoutesttomakesureyourvectoriscorrect?

How to test for correct product...

A B C A C

A C

Ligase

A C

Parent

Correct A + C

Correc

t

10

How to test for correct product...

A B C A C

A C

Ligase

A C

A B C

+

A B C

+

Parent

Correct A + C

Correc

t Pare

nt

How to test for correct product...

A B C A C

A C

Ligase

A C

A B C

+

A B C

+

Parent

C + D Correct

Correc

t Pare

nt

D

D

Be sure the expected fragment is easy to see and evaluate

(e.g., 0.3 to 2 kb)

How to test for correct product...

A B C A C

A C

Ligase

A C

A B C

+

A B C

+

Parent

A + D Correct

D

D

Correc

t Pare

nt

E

How to test for correct product...

A B C A C

A C

Ligase

A C

A B C

+

A B C

+

Parent

A + D

Correct

Correc

t Pare

nt

D

D E

E + F

Correc

t Pare

nt

Why not use E + D?

What will E+F find that A+D misses?

F F

F F

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GoingfromUnderstandingtoSolu6ons

‐Exploi6ngUnderstandingofHRforgene6cengineering

Mod1Overview:MethodsandLogic

‐Logicforstepssofar‐Strategyforanalysis