TOOLS FOR STUDYING SIGNALING IN IMMUNE CELLS

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TOOLS FOR STUDYING SIGNALING IN IMMUNE CELLS

• CHUJOR S.N. CHUJOR,
• ALBERT EINSTEIN HIGH SCHOOL
• MENTOR RAJAT VARMA,
• LABORATORY OF CELLULAR AND MOLECULAR IMMUNOLOGY,
  NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS
  DISEASES, NIH

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BACKGROUND

• Several molecules play key roles in the
  initiation and orchestration of immune responses
  against invasive foreign substances or infectious
  organisms.
• The signaling pathways that mediate this cellular
  event involve interaction between receptors and
  signaling molecules.

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CYTOKINE SIGNALING PATHWAY
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OVERALL GOAL

• To investigate cell surface dynamics,
  signaling, and trafficking of cytokine receptors
  
• To study these properties in various cell types,
  receptor molecules would be tagged to GFP and its
  variants.

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SPECIFIC OBJECTIVES

• Determination and analysis of nucleotide
  structure of selected cytokines and receptor
  molecules
• Generation of Mutated RFP-containing Constructs
  For Enhanced Photo-stability
• Generation and Verification of Full-Length cDNA
  for Selected Cytokine/Receptor Molecules
• Design of Customized Primers for Sequencing and
  PCR work

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EXPERIMENTAL DESIGN - I

• Verification of cDNAs for Cytokine Receptors and
  Janus Kinases for Studying Cytokine Signaling in
  Immune Cells.
• Transformation of Competent Bacteria Cells, DH-5a
  T1, with Recombinant Plasmid DNAs
• Isolation and Expansion of Colonies Harboring
  Drug-resistant Constructs of Interest
• Extraction and Purification of DNA
• Sequencing and Computer-aided Analysis of
  Nucleotide Structure of Selected
  Cytokine/Receptor Molecules
• Gel Electrophoresis and PCR (Amplification) of
  DNAs

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EXPERIMENTAL DESIGN - II

• Primer Design
• Sequencing Primers
• PCR Primers
• Mutagenesis Primers
• Cloning strategies
• Mutagenesis of p65ENDOp-TagRFP-p65-C1 Construct
• Transformation of DH-5a T1 Cells with Mutated
• p65ENDOp-TagRFP-p65-C1 Construct
• Extraction, Purification, and Sequencing of
  Mutated p65ENDOp-TagRFP-p65-C1 Construct

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RECOMBINANT PLASMID DNA CLONING DATA

• Verification of cDNA Sequences
• cDNA Size (Bp) Full-Length Sequence Status
• Previous-08 Current-09
• Jak 1 2365 No Yes
• Jak 2 4013 No Yes
• Jak 3 3749 No Yes
• IL-2Ra 1420 Yes Yes
• IL-2Rß 2678 No Yes
• IL-2R? 1800 Yes Yes
• IL-4Ra 2043 No Yes
• IL-15Ra 1465 Yes Yes

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RECOMBINANT PLASMID DNA CLONING DATA Sequencing
Primers for Cytokines/Receptors - I Jak1-1Kb 5
TTC AGA AAA GCA GCC AAC AAC AG 3 Jak2-796 5
ACA GAT TTC GCA GAT TCA TTC AGC 3 Jak2-2591 5
CCT AGG GTT TTC TGG TGC TTT TG AG
3 Jak2-2688R 5 TCA TAG CGG CAC ATC TCC ACA CT
3 Jak3-1010 5 CCT GAG GCG CTG TCT TTC GTG
3 Jak3-1791 5 TCA TGG TGC AGG AAT TTG TGT ATC
3 Jak3-2688 5 AGA GCC TGC GGT TGG TGA TGG AG 3
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RECOMBINANT PLASMID DNA CLONING DATA Sequencing
Primers for Cytokines/Receptors-II IL-2Rß-940 5
TTG GGC CAT GGC TGA AGA CA 3 IL-2Rß-1715 5
GGC CCA AGA TTC AGT CCA CCT AA 3 IL-2Rß-2289 5
GAG GGG TTG GGA AGG GTC ATC AGT
3 IL-4Ra -SP6 SP6 Sequencing Primer IL-4Ra-936
5 AGC GCC TTC CAC TGG GGG GTC AC
3 IL-4Ra-1839 5 GGC CGG C TG ACC CCC ATT CTT 3
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RECOMBINANT PLASMID DNA CLONING DATA PCR Primers
for Cytokine/Receptor Molecules Jak1-Sal1-F 5
TAT CGT CGA CCC ACG CGT CCG ATG AAC
3 Jak1-SacII-R 5 CAT CCG CGG TAA AAG TGC TTC
AAA TCC TCC AA 3 Jak2-Sal1-F 5 ACG CGT CGA CAT
GGG AAT GGC CTG C 3 Jak2-SacII-R 5 GAT CCG CGG
CGC AGC TAT ACT GTC C 3 Jak3-Sal1-F 5 ACG CGT
CGA CAT GGC ACC TCC AAG TG 3 Jak3-SacII-R 5 AGT
CCG CGG TCC GGG TCT TCC ACG 3 IL-2Ra-Sal1-F 5
TAT CGT CGA CGC CAG GAA GAT GGA GC
3 IL-2Ra-SacII-R 5 AAG AGC AGA AGA ACC ATC CCG
CGG ATC 3 IL-2R?-Sal1-F 5 ACG CGT CGA CAT GTT
GAA ACT ATT ATT GTC 3 IL-2R?-SacII-R 5 TCT CCG
CGG GGC TTC CGG CTT CAG 3
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RECOMBINANT PLASMID DNA CLONING DATA Tag RFP
DNA Sequence (Published) ATGGTGTCTAAGGGCGAAGAGCT
GATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGAACA
ACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGC
ACCCAGACCATGAGAATCAAGGTGGTCGAGGGCGGCCCTCTCCCCTTCGC
CTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAGAACCTTCATCA
ACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGC
TTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGCTGACCGC
TACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGA
TCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACA
CTCGGCTGGGAGGCCAACACCCCGCTGACGAGATGCTGTACCGGCGGCCT
GGAAGGCAGAAGCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGA
TCTGCAACTTCAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTC
AAGATGCCCGGCGTCTACTATGTGGACCACAGACTGGAAAGAATCAAGGA
GGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGAT
ACTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATTGA
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RECOMBINANT PLASMID DNA CLONING
DATA Mutagenesis PCR Primers for RFP
Sequence S162 T F new 5 CTG GAA GGC AGA ACC
GAC ATG GCC CTG 3 Tm 66.6oC S162 T R
new 5 CAG GGC CAT GTC GGT TCT GCC TTC CAG
3 Tm 66.6oC PA RFP F 5 TCC CCG
CGG ATG GTG TCT AAG GGC GAA GAG 3 Tm
68.9oC Pa RFP R 5 TCT ACC GGT TCA ATT AAG
TTT GTG CCC CAG 3 Tm 61.9oC
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RESTRICTION ENZYME ANALYSIS OF p65ENDOp-TagRFP-p65
-C1 VECTOR
1 2 3
Bp
6000
3998 Bp
3000
1000
735 Bp
750
Lane 1 1 Kb DNA Maker Lane 2 Undigested
Vector Lane 3 Age 1 vs. Not 1 Digestion
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PCR AMPLIFICATION OF Tag RFP DNA SEGMENT
Lane 1 2
Bp
6000
3000
1000
750
735 Bp
Lane 1 1 Kb DNA Maker Lane 2 RFP-Primer
Fragment
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PCR AMPLIFICATION OF p65ENDOp-TagRFP-p65-C1 VECTOR
Lane 1 2 3
Bp
7600 Bp
6000
3000
1000
Lane 1 1 Kb DNA Maker Lane 2 S162TF vs. S162TR
Primers Sample 1 Lane 3 S162TF vs. S162TR
Primers Sample 2
500
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RECOMBINANT PLASMID DNA CLONING DATA Generation,
Isolation, and Analysis of Bacteria Colonies
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RECOMBINANT PLASMID DNA CLONING
DATA Verification of DNA Sequences Tag RFP
GCTGTACCGGCGGCCTGGAAGGCAGAAGCGACATGGCCCTGAAGCTCG
TGGGCGGG ST1 Clone GCTGTACCGGCGGCCTGGAAGGCAGAACCG
ACATGGCCCTGAAGCTCGTGGGCGGG TAG RFP ...
LYPADGGLEGRSDMALKLVGG. ST1 CLONE ...
LYPADGGLEGRTDMALKLVGG..
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NEXT STEP 1 GENERATION OF FUSION
CONSTRUCTS Jak 1 Tag RFP (S162T) Jak 2
Tag RFP (S162T) Jak 3 Tag RFP
(S162T) IL-2Ra Tag CFP IL-2Ra Tag
YFP IL-2Rß Tag CFP IL-2Rß Tag
YFP IL-2R? Tag YFP IL-2R? Tag
RFP (S162T) IL-4Ra Tag CFP
IL-4Ra Tag YFP IL-15Ra Tag CFP
IL-15Ra Tag YFP
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NEXT STEP 2 TRANSFECTION OF CELL TYPES WITH
FUSION CONSTRUCTS NEXT STEP 3
BIOCHEMICAL AND BIOPHYSICAL STUDIES OF CELL
SURFACE DYNAMICS, SIGNALING, AND TRAFFICKING OF
CYTOKINE RECEPTORS
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SUMMARY

• Tools for studying cell surface dynamics,
  signaling, and trafficking of cytokine receptors
  are being developed
• cDNA sequences for all selected cytokine
  receptors have been verified
• p65ENDOp-TagRFP-p65-C1 Vector carrying mutated
  RFP Gene has been constructed and verified by DNA
  sequencing
• PCR and Sequencing Primers for all selected
  cytokine receptors have been designed and
  synthesized.

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ACKNOWLEDGEMENTS

• Gratefully acknowledge
• HHMI/NIH/MCPS for giving me this great research
  opportunity
• LCMI (NIAID), Dr. Schwartz, for warm welcome
• My Mentor, Dr. Rajat Varma, for professional
  advice and guidance
• Colleagues in our Lab (Drs. T. Crites and K.
  Padhan) for friendly interaction and helpful
  exchange of ideas
• Dr. Clare Waterman-Storer for help with some
  reagents
• Ms. Jill Latchana for effective communication and
  coordination
• Mr. James Fernandez and Mr. Damian DiCamillo for
  their support and encouragement