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