PROJECT OUTLINE

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PROJECT OUTLINE

• Identifying cd3d interactors by membrane yeast
  two hybrid system
• Structure function studies in cd3d interactors.
• Generating cd3d deficient T lymphocytes
• shRNA Mediated Expression Arrest-in cell lines
• Recombineering Generating targeting construct
  KO mouse and KO cell line
• Studying TCR signaling in cd3d -/- systems

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DUAL MEMBRANE SYSTEM
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BAIT-CD3d VECTOR
PREY-LIBRARY VECTOR
C
membrane
N
C
N
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LIBRARY SCREENING
C
membrane
N
C
N
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LIBRARY SCREENING
Figure Positive colonies on selective
plates(-LTHA) and corresponding b-galactosidase
filter lift-off assay
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PUTATIVE INTERACTORS(IDENTIFIED SO FAR)
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MAMMALIAN STRATEGY
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Ligationo/n 16C
Colony PCR
Colony PCR
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Hind3-Xho1 digest Pop out insert 525bp
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TRANSFECTION OF 293T CELLS
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TRANSFECTION EFFICIENCY

• pcDNAGFP transfected 293T cells

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CELL LYSIS and IMMUNOPRECIPITATION

• 48 hr after transfection lyse the cells using
  TX100 Lysis Buffer

• Save 50 ml of lysate and use the rest for
• immunoprecipitation with either aHA OR
• a-cMyc

• For HA immunoprecipitation directly add aHA
  beads on the lysed cells.
• For Myc immunoprecipitation First immobilize
  acMyc on sepharose beads
• Then use the immobilized antibody for the
  immunoprecipitation.
• After immunoprecipitaion save the IPs at -20
  degrees.

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SDS GEL and WESTERN BLOT

• Pour the seperating and stacking gels
• Samples add SDS loading buffer and boil 5 min
• Load the samples
• Run the gel 100V(const) for 2 hrs.
• Transfer the bands to membrane. (100V for 1hr)
• Block the membrane(PBSTMilk/BSA) o/n
• Incubate with antibody
• a Myc peroxidase(1-22000)
• OR a HAPeroxidase (4-202000)
• Wash w PBST
• Film in dark room

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RESULTS OF LYSATES
a-HA
a-Myc
25KDa
30KDa
a-Myc
a-Myc
25KDa
25KDa
a-Myc
CD81(26KDa) HA(1KDa) CD3D(19KDa)MYC(1KDa)
25KDa
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MAMMALIAN STRATEGY
Sfi1
pCMV
Sfi1
HA
Sfi1
MCS
pHA-Mex (4050BP)
KanR
Sfi1 digest
Sfi1 digest

• pHA-Mex midiprep
• Confirmation
• Sfi1 digest for ligation

• cDNAs miniprep
• Sfi1 digest for ligation

Ligation
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• cDNAs miniprep all done
• Sfi1 digest for ligation(5hrs)
• 3I1- CD81
• 3C1- BCAP31
• 1G6- ICAM2
• 1G4- RAG1AP1
• 3A7-TXN

Gel extraction
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• pHAMex midi
• Sfi1 digest for ligation(o/n)

Gel extraction
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Ligations 16C o/n
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Miniprep-Sfi1 digest
Further digest to confirm
?
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Miniprep-Sfi1 digest
pHAMex-3
3 3A7aTXN
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Miniprep-Sfi1 digest
pHAMex-G6
pHAMex-G4
G4-RAG1AP1
G6-RAG1AP1
Further digest to confirm Transfection for coIP
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Miniprep-Sfi1 digest
pHAMex-G4
pHAMex-G6
G4-RAG1AP1
G6-ICAM2
Further digest to confirm Transfection for coIP
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pHAMex-CD811
pHAMex-BCAP31
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RECOMBINATION TARGETING PLASMID (Step1)
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RECOMBINATION TARGETING PLASMID (Step2)
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RECOMBINATION TARGETING PLASMID (Step3)
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Direct cloning didnt work TA cloning
New PCR for TA cloning final extension 20 min
PCR purification
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InsTACloning Kit-FermentasLigation 3hr on bench
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Colony PCR using previous primers
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TA-R3 and T1-R5 miniprepSal1Xho1 digest
R3
R5
3 4 5
3 4 5
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shRNA MEDIATED EXPRESSION ARREST
Detect change in GFP ..need stable GFP
expressing 2B4
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shRNA MEDIATED EXPRESSION ARREST
CaCl2 transfection
pSM2-EGFP IRES2EGFP pSM2-cd3D1 pSM2-cd3D2
Retroviruses
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Negative controlPCR of scrambler oligo
PCR purification XhoR1 digest Clone into LMP
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2B4-STABLE GFP
EP-15ug circular pIRES2EGFP
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