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Characterizing RNF20 and RNF40 in Class Switching of B CellsCathy Tie
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Introduction• Somatic hypermutation and class switch recombination
(CSR) contribute to effective immune response
• B cells produce antibodies to clear pathogens and toxins
• CSR induced by activation-induced cytidine deaminase(AID) gene at switch regions within the Ig locus
• Different antibody isotypes have distinct effector functions
• B cells switch from class IgM to IgA
• This study looks at proteins RNF20 and RNF40 in the process of CSR
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Antibody Isotypes• The antibody class is
the constant region in
an antibody that can
only be replaced with
other constant regions
by CSR.
• It can change the
antibody effector
functions without
changing the antigen
binding site.
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Class Switch Recombination (CSR)• B cells switch from IgM to IgA
• Detection and processing of the mutated residues by base excision repair generates double-stranded DNA breaks (DSBs) at switch regions
• DSBs cause B cells to signal a damage response to mend the resultant breaks
• CSR removes portions in the antibody heavy chain locus from the chromosome
• The surrounding gene segments rejoin in the heavy chain to generate a different antibody gene that produces antibody of a different isotype
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Durandy A (2003). "Activation-induced cytiddeaminase: a dual role in class-
switch recombination and somatic hypermutation". Eur. J. Immunol. 33 (8):
2069–73.
Activation-Induced CytidineDeaminase Protein (AID)
• Recent discovery of AID furthered our knowledge of somatic hypermutation and class switch recombination (CSR)
• Mice and humans deficient in AID are incapable of somatic hypermutation and CSR
• AID is the only B cell specific protein that is required for both of these processes
• AID initiates antibody diversification by deaminating cytidineswithin switch regions for class switch recombination
• Current research revolves around delineating the class switch recombination processes including the DNA repair proteins that repair the AID-induced DNA lesions
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To what extent are RNF20 and RNF40 crucial proteins in the class switch recombination process in B cells?
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Procedures
• Extract mRNA from B cell line CH12F3-2 and construct cDNA library using reverse transcriptase
• Amplify the RNF20 and RNF40 cDNAs from the cDNAlibrary using the Polymerase Chain Reaction (PCR) technique with a specific primer pair
• Primers mark the target sequence to be amplified
• The size of the DNA molecule will be determined using the agarose gel method
• Size will be compared to information indicated in the National center for Biotechnology Information
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Polymerase –Chain Reaction (PCR)• Denaturation heats up the
reaction for melting of the DNA template by disrupting the hydrogen bonds between complementary bases
• Yields single-stranded DNA molecules
• Primers mark the ends of target sequence in the annealing stage
• GADPH primers were used as the control
• RNF20 ‘UTR, RNF40 ‘TUR, and RNF20/40 clone with extensions were tested
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Polymerase –Chain Reaction (PCR)• Each primer binds to one DNA
strand produced during denaturation
• Temperature is raised to replicate the DNA strands by the use of Phusion
• Phusion DNA polymerase facilitates the binding and joining of the complementary nucleotides that are free in solution (dNTPs), thereby synthesizing new double stranded DNA molecules
• Phusion DNA polymerase functions in the 5’ to 3’ direction
• Free nucleotides in the solution are only added to the 3’ end of the primers constructing the complementary strand of the targeted DNA sequence
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Electrophoresis
PCR products (DNA molecules) move from top (negative charge) to bottom (positive charge) between the spaces in the agarose gel. Smaller molecules tend to travel faster than larger molecules.
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Procedures cont’d• PCR purification cleans the DNA of the gene
• RNF20 and RNF40 PCR products will be ligated with a specifically cut drug resistant vector
• Parts of RNF20 and RNF40 will be digested by restriction enzyme to fit with vector
• PCR product and vector combined by ligation, forming a plasmid that is resistant to the drug ampicillin
• Pasmid placed into bacteria for continual generation
• RNF20 and RNF40 return to B cell line, observe the effect on CSR
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Objectives• Test control primers (GADPH) with Taq
• Test the combinations of cDNA oligo DT and hexamer with various primers
• Test different primers and Phusion in gradient PCR with cDNA RNF20 5’/3’UTR
RNF20 Clone
RNF40 5’/3’UTR
RNF40 Clone
• Understand the importance of the ubiquitin system in antibody diversification and DNA damage response
• Use a flow cytometry assay to determine whether overexpression of removal of RNF20/RNF40 impacts CSR
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Summary of Hypotheses•
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GADPH PrimersThrough electrophoresis analysis, the control reveals successful bands at 350 – 400 base pairs
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ladder GADPH Primers with cDNA
GADPH and RNF20/40 ‘UTR PrimersElectrophoresis analyses of cDNA, generated from oligo dT-mediated reverse transcription with GAPDH and RNF20 and RNF40 ‘UTR primers. RNF20 and RNF40 ‘UTR primers result in streaking with the absence of DNA bands
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RNF20/40 ‘UTR Primers w/ 4+ and 7+ cDNATwo samples of cDNA, generated from oligo dT-mediated reverse transcription, were tested with
RNF20/40 ‘UTR primers and Taq; no bands present
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ladder
ladder
RNF40 ‘UTR with 4+ RNF40 ‘UTR with 7+
RNF20 ‘UTR with 4+ RNF20 ‘UTR with 7+
RNF20/40 ‘UTR and Clone PrimersConsistent cDNA sample was tested with the four primers above. Clone primers consisted of
extensions which will be later digested by enzymes for ligation with vector; no bands present
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RNF40 ‘UTR with 4+ RNF40 clone with 4+
RNF20 ‘UTR with 4+ RNF20 clone with 4+
RNF20/40 ‘UTR and Clone Primers with hexamer cDNASame primers were tested with oligo DT and random hexamer cDNA samples. Phussion
also proved to be more effective than Taq at PCR. No bands present.
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Standard Curve for 100 base pairs (bp) Ladder
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• Control GAPDH primers resulted in
DNA bands
• From slide 11, bands by GAPDH with
cDNA 4+ and 7+ migrated 14mm and
13.5mm from the well
• Standard 100 bp curve shows that they
are 350 bp and 400 bp respectively
• 4+ DNA molecule is smaller than the
7+ molecule
• GADPH primers succeeded in
expressing GADPH genes, as NCBI
states that the correct size is 350 bp
• The -1kp ladder curve will be used to
measure the size of the RNF20 and
RNF40 amplified molecules
Discussion• Problems remain in the Polymerase Chain Reaction (PCR)
method, specifically in the function of primers Inefficiency of primers
Buffer or temperature range was optimal
Genes were expressed at very low levels
• Conclusion Primers below are not effective in expressing RNF20 and RNF40
• Require more effective RNF20 and RNF40 primers in combination with Phusion in PCR
• Successful PCR products, shown through electrophoresis analyses, will be cut and ligated with a vector to form a plasmid resistant to ampicillin C
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RNF20 5’UTR: ATGTCAGGAATTGGAAATAAAAG
RNF20 3’UTR: GCCAATGTAGATGCGATGGAA
RNF20 Clone F: GAACGGATCCCACCATGTCAGGAATTGGAAATAAAAG
RNF20 Clone R: TGTCGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTATGATCCGCCAATGTAGATGCGATGGAA
RNF40 5’UTR: ATGTCTGGCCTCAGCAACA
RNF40 3’UTR: GCTGATGTACACACGGTGG
RNF40 Clone F: GAACGCTAGCCACCATGTCTGGCCTCAGCAACA
RNF40 Clone R: TGTCGCGGCCGCTCAAGCGTAATCTGGAACATCGTATGGGTATGATCCGCTGATGTACACACGGTGG
Discussion and Future Steps• Drug-resistant plasmids will be exponentially generated
• Overexpression of proteins will affect CSR
• Protein depleted cells should exhibit no defects in proliferation, as reduced CSR is only due to the functions of RNF20 and RNF40
• The effects of the proteins’ knockdown by immunofluorescence will show the roles of the proteins
• Roles involved DNA damage response (DDR) will translate to knowledge about cancer classification and development
DDR protects against tumour progression spreading to daughter cells
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Conclusion
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Acknowledgements
• Thank you to my mentors, Dr. Martin and Shaliny R., at the Martin Laboratory at the University of Toronto for providing guidance and laboratory equipment for this project
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