Name: assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes
Uploaded: 2011-11-01T13:00:00
Description: Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Den pMSCV-Aid-I-Thy1.1 og pKat2 vektorer var en slags gave fra DG Schatz og pVSV-G, pRSV-Rev, og pMDLg / pRRE vektorer var en slags gave fra BR Cullen.

1. Forbereder prøver og celler <ol><li> Utfør en medium skala utarbeidelse av DNA (midiprep) av overekspresjon eller shRNA inneholder konstruerer og retrovirale emballasjen vektor pKat2 eller lentiviral emballasjen vektorer pVSV-G, pMDLg / pRRE og pRSV-Rev 25. Bruk 6 mikrogram DNA for hver konstruere og enten 4 mikrogram pKat2 (for retrovirale infeksjoner) eller 1,5 mikrogram DNA for hver av de pVSV-g, pMDLg / pRRE og pRSV-Rev emballasje vektorer (for lentiviral infeksjoner). </li><li> Klargjør Bosc 23 m...

<ol>
	<li>Di Noia, J. M., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibody+somatic+hypermutation.">Molecular mechanisms of antibody somatic hypermutation.</a> Annu. Rev. Biochem. 76, 1-22 (2007).</li><li>Peled, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+somatic+hypermutation.">The biochemistry of somatic hypermutation.</a> Annu. Rev. Immunol. 26, 481-511 (2008).</li><li>Longerich, S., Basu, U., Alt, F., Storb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+in+somatic+hypermutation+and+class+switch+recombination.">AID in somatic hypermutation and class switch recombination.</a> Curr. Opin. Immunol. 18, 164-174 (2006).</li><li>Bardwell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+somatic+hypermutation+and+reduced+class-switch+recombination+in+exonuclease+1-mutant+mice.">Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.</a> Nat. Immunol. 5, 224-229 (2004).</li><li>Martomo, S. A., Yang, W. W., Gearhart, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Msh6+but+not+Msh3+in+somatic+hypermutation+and+class+switch+recombination.">A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination.</a> J. Exp. Med. 200, 61-68 (2004).</li><li>Phung, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hypermutation+at+G+and+C+nucleotides+in+immunoglobulin+variable+genes+from+mice+deficient+in+the+MSH2+mismatch+repair+protein.">Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.</a> J. Exp. Med. 187, 1745-1751 (1998).</li><li>Rada, C., Ehrenstein, M. R., Neuberger, M. S., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+spot+focusing+of+somatic+hypermutation+in+MSH2-deficient+mice+suggests+two+stages+of+mutational+targeting.">Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.</a> Immunity. 9, 135-141 (1998).</li><li>Rada, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+isotype+switching+is+inhibited+and+somatic+hypermutation+perturbed+in+UNG-deficient+mice.">Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.</a> Curr. Biol. 12, 1748-1755 (2002).</li><li>Delbos, F., Aoufouchi, S., Faili, A., Weill, J. C., Reynaud, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+the+sole+contributor+of+A/T+modifications+during+immunoglobulin+gene+hypermutation+in+the+mouse.">DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.</a> J. Exp. Med. 204, 17-23 (2007).</li><li>Masuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+a+limiting+factor+for+A:T+mutations+in+Ig+genes+and+contributes+to+antibody+affinity+maturation.">DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.</a> Eur. J. Immunol. 38, 2796-2805 (2008).</li><li>Lim, D. S., Hasty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+mouse+rad51+results+in+an+early+embryonic+lethal+that+is+suppressed+by+a+mutation+in+p53.">A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.</a> Mol. Cell. Biol. 16, 7133-7143 (1996).</li><li>Xiao, Y., Weaver, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+targeted+deletion+by+Cre+recombinase+demonstrates+the+requirement+for+the+double-strand+break+repair+Mre11+protein+in+murine+embryonic+stem+cells.">Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.</a> Nucleic. Acids. Res. 25, 2985-2991 (1997).</li><li>Zhu, J., Petersen, S., Tessarollo, L., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+Nijmegen+breakage+syndrome+gene+NBS1+leads+to+early+embryonic+lethality+in+mice.">Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.</a> Curr. Biol. 11, 105-109 (2001).</li><li>Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mRad50+causes+embryonic+stem+cell+lethality,+abnormal+embryonic+development,+and+sensitivity+to+ionizing+radiation.">Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 7376-7381 (1999).</li><li>Pavri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation-induced+cytidine+deaminase+targets+DNA+at+sites+of+RNA+polymerase+II+stalling+by+interaction+with+Spt5.">Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.</a> Cell. 143, 122-133 (2010).</li><li>Lee-Theilen, M., Matthews, A. J., Kelly, D., Zheng, S., Chaudhuri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CtIP+promotes+microhomology-mediated+alternative+end+joining+during+class-switch+recombination.">CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.</a> Nat. Struct. Mol. Biol. 18, 75-79 (2011).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+exosome+targets+the+AID+cytidine+deaminase+to+both+strands+of+transcribed+duplex+DNA+substrates.">The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates.</a> Cell. 144, 353-363 (2011).</li><li>Yabuki, M., Fujii, M. M., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MRE11-RAD50-NBS1+complex+accelerates+somatic+hypermutation+and+gene+conversion+of+immunoglobulin+variable+regions.">The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.</a> Nat. Immunol. 6, 730-736 (2005).</li><li>Sale, J. E., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TdT-accessible+breaks+are+scattered+over+the+immunoglobulin+V+domain+in+a+constitutively+hypermutating+B+cell+line.">TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line.</a> Immunity. 9, 859-869 (1998).</li><li>Cumbers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+iterative+affinity+maturation+of+antibodies+in+vitro+using+hypermutating+B-cell+lines.">Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.</a> Nat. Biotechnol. 20, 1129-1134 (2002).</li><li>Papavasiliou, F. N., Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-regulated+DNA+double-stranded+breaks+in+somatic+hypermutation+of+immunoglobulin+genes.">Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.</a> Nature. 408, 216-221 (2000).</li><li>Parsa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+mutates+a+non-immunoglobulin+transgene+independent+of+chromosomal+position.">AID mutates a non-immunoglobulin transgene independent of chromosomal position.</a> Mol. Immunol. 44, 567-575 (2007).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+instability+of+V+region+hypermutation+in+the+Ramos+Burkitt's+lymphoma+cell+line.">Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.</a> Int. Immunol. 13, 1175-1184 (2001).</li><li>Ukai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a:T+mutations+is+dependent+on+cellular+environment+but+independent+of+mutation+frequency+and+target+gene+location.">Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.</a> J. Immunol. 181, 7835-7842 (2008).</li><li>Dull, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+class+switching+of+an+IgM++B+lymphoma+clone+CH12F3+to+IgA++cells.">High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells.</a> Int. Immunol. 8, 193-201 (1996).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+activation-induced+cytidine+deaminase+(AID),+a+novel+member+of+the+RNA-editing+deaminase+family+in+germinal+center+B+cells.">Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.</a> J. Biol. Chem. 274, 18470-18476 (1999).</li></ol>

Som diskutert tidligere, har cellelinje modeller for antistoff diversifisering blitt et populært utgangspunkt for å identifisere nye proteiner som påvirker ulike trinnene i løpet av antistoff diversifisering. Vi presenterer her en metode for å bruke viral infeksjon enten knock-down eller overuttrykker proteiner i Ramos B-celle linje og deretter undersøke virkningen på SHM. For disse studiene benytter vi både WT Ramos og AID hi Ramos celler. WT cellene kan brukes til å stu...

Forslag til reagenser - de fleste av disse kan erstattes med tilsvarende produkter fra andre leverandører. <table border="1" ><tbody><tr><th> Navn på reagens </th><th > Firma </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td > 6-godt klar TC-behandlede plater </td><td > Corning </td><td> 3516 </td><td ></td></tr><tr><td > 10 mL BD Luer-Loksyringes </td><td > BD Medical </td><td > 309604 </td><td ></td></tr><tr><td> 24-godt klar TC-behandlede plater </td><td > Corning </td><td > 3526 </td><td></td></tr><tr><td > 96-godt klar flat bunn polystyren TC-behandlet mikroplater </td><td > Corning </td><td > 3596 </td><td ></td></tr><tr><td > 100 mm TC-behandlede kulturen retter </td><td > Corning </td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc sprøyte filtre, 0,45 mikrometer </td><td > Pall Life Sciences </td><td > 4604 </td><td ></td></tr><tr><td > Agar </td><td > Teknova </td><td > A7777 </td&gt; <td ></td></tr><tr><td > Agarose </td><td > GeneMate </td><td> E-3120-500 </td><td ></td></tr><tr><td > Ampicillin </td><td> Sigma </td><td > A0166 </td><td> 100 mg / ml i vann </td></tr><tr><td > BD FACSCanto II flowcytometer </td><td> BD Biosciences </td><td ></td><td > eller lignende </td></tr><tr><td> BD Falcon rund bunn polystyren rør </td><td> BD Biosciences </td><td > 352054 </td><td > for FACS </td></tr><tr><td > Bosc 23 celler </td><td > ATCC </td><td > CRL-11270 </td><td ></td></tr><tr><td > CO 2 inkubator i stand til 37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (Dulbecco modifiserte Eagle medium) </td><td > Sigma </td><td> D6429 </td><td ></td></tr><tr><td > FBS (fetal bovint serum) </td><td > Gemini Bio-produkter </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-rotte CD90/mouse CD90.1 antistoff </td><td > BioLegend </td><td > 202503 </td><td > FITCα-Thy1.1 </td></tr><tr><td > FuGENE 6 Transfeksjon Reagens </td><td > Roche </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES bufferløsning </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi DNA polymerase </td><td > KAPA Biosystems </td><td > KK2101 </td><td ></td></tr><tr><td > LB buljong (lysogeny kjøttkraft - Luria) Powder </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > MISSION TRC shRNA bakteriell glyserol lager </td><td > Sigma </td><td ></td><td> shRNA vektorer </td></tr><tr><td > NCS (nyfødt kalv serum) </td><td > Gemini Bio-produkter </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (fosfatbufret løsning) </td><td > Invitrogen </td><td> 70011 </td><td > fortynnes til 1x i vann </td></tr><tr><td > PE α-human IgM antistoff </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (penicillin-streptomycin-glutamin løsning) </td><td > Gemini Bio-Produkter </td><td > 400-110 </td><td ></td></tr><tr><td> Polybrene (hexadimethrine bromide) </td><td > Sigma </td><td > 107689 </td><td > 10 mg / ml i vann </td></tr><tr><td > PureYield Plasmid Midiprep System </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Puromycin </td><td> Sigma </td><td > P8833 </td><td > 250 mikrogram / ml i vann </td></tr><tr><td > QIAquick gel utvinning kit </td><td > Qiagen </td><td > 28706 </td><td ></td></tr><tr><td> Ramos (RA 1) celler </td><td > ATCC </td><td > CRL-1596 </td><td ></td></tr><tr><td > RPMI-1640 medium </td><td > Sigma </td><td > R8758 </td><td ></td></tr><tr><td > Hevet II </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR FAST qPCR kit </td><td > KAPA Biosystems </td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNA Polymerase </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > Topo TA Cloning kit </td><td > Invitrogen </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > Wizard SV Genomisk DNA rensing system </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-Bromo-4-klor-3-indoyl-β-D-galatopyranoside] </td><td > Growcells </td><td > C-5687 </td><td > 40 mg / ml i DMSO </td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

B-celler starter sitt liv med lav affinitet antistoffer genereres av V (D) J rekombinasjon. Men når det oppdages et patogen, er den variable (V) region av et immunoglobulin (Ig) genet mutert omtrent 100.000 ganger mer enn resten av genom gjennom somatiske hypermutation (SHM), som resulterer i høy affinitet antistoffer 1,2. I tillegg produserer klasse bytte rekombinasjon (CSR) antistoffer med forskjellige effektor funksjoner avhengig av typen av immunrespons som er nødvendig for en bestemt patogen. Både CSR og SHM er initiert av aktivisering-indusert cytidin deaminase (AID), som deaminates cytosin rester i DNA til å produsere uracils. Disse uracils behandles av feilutsatt former for reparasjon pathways, til slutt fører til mutasjoner og rekombinasjon 1-3. Vår nåværende forståelse av molekylære detaljene i SHM og CSR kommer fra en kombinasjon av studier i mus, primære celler, cellelinjer, og celle-fRee eksperimenter. Musemodeller forblir gullstandarden med genetisk knockouts viser kritiske roller for mange reparasjon faktorer (f.eks Ung, Msh2, Msh6, Exo1, og polymerase η) 4-10. Men ikke alle gener er mottagelig for knockout studier. For eksempel, knockouts av flere dobbel tråd pause reparasjons proteiner er embryonically dødelig eller forringe B-celle utvikling 11-14. Dessuten, noen ganger spesifikk funksjon av et protein i SHM eller CSR kan maskeres av mer global defekter forårsaket av knockout. I tillegg kan siden eksperimenter i mus være lange, endre uttrykket av individuelle gener i celle linjer har blitt et stadig mer populært første skritt for å identifisere og karakterisere kandidat gener 15-18. Ramos - en Burkitt lymfom cellelinje som konstitutivt gjennomgår SHM - har vært et populært celle-line modell for å studere SHM 18-24. En fordel med Ramos celler er at de har en innebygd praktisk semi-Kvantitative mål på SHM. Wild typen celler uttrykker IgM, og som de plukker opp mutasjoner, noen av mutasjonene knock out IgM uttrykk. Derfor analysere IgM tap av fluorescens-aktivert celle skanning (FACS) gir en rask lese-out for nivået på SHM. En mer kvantitativ måling av SHM kan fås ved direkte sekvensering antistoffet gener. Siden Ramos celler er vanskelig å transfektere, produserer vi stabile derivater som har økt eller senket uttrykk for en individuell genet ved å infisere celler med retrovirale eller lentiviral konstruksjoner som inneholder enten en overekspresjon kassett eller en kort hårnål RNA (shRNA), henholdsvis. Her beskriver vi hvordan vi infisere Ramos celler og deretter bruke disse cellene til å undersøke hvilken rolle spesifikke gener på SHM (Figur 1).

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

Vi beskriver hvordan du utfører retrovirale eller lentiviral infeksjoner i overekspresjon eller shRNA inneholder konstruerer i den menneskelige Ramos B-celle linje og hvordan måle somatiske hypermutation i disse cellene.

Vurdering av Somatiske Hypermutation i Ramos B celler etter Overuttrykte eller knockdown av spesifikke gener

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

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