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

PMSCV-AID-I-Thy1.1 и pKat2 векторы были своего рода подарок от Д. Г. Шац и pVSV-G, pRSV-Rev, и pMDLg / pRRE векторы были своего рода подарок от BR Каллен.

1. Подготовка образцов и клетки <ol><li> Выполнить средний подготовки масштаба ДНК (midiprep) из гиперэкспрессия или shRNA содержащих конструкции и упаковки ретровирусных вектор pKat2 или лентивирусов векторов упаковки pVSV-G, pMDLg / pRRE и pRSV-Rev 25. Используйте 6 мкг ДНК для каждой конструкции и либо ...

<ol>
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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. 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Как обсуждалось ранее, клеточная линия моделей для антител диверсификации стали популярным отправной точкой для выявления новых белков, которые влияют на различные шаги в процессе антител диверсификации. Мы приведем здесь способ использования вирусной инфекции либо нокдауна или гип...

Предлагаемые реагенты - большинство из них могут быть заменены аналогичными продуктами от других производителей. <table border="1" ><tbody><tr><th> Название реагента </th><th > Компания </th><th> Номер по каталогу </th><th> Комментарии </th></tr><tr><td > 6-подальше TC обработанные пластины </td><td > Гранулирование </td><td> 3516 </td><td ></td></tr><tr><td > 10 мл BD Luer-Loksyringes </td><td > BD Медицинские </td><td > 309604 </td><td ></td></tr><tr><td> 24-подальше TC обработанные пластины </td><td > Гранулирование </td><td > 3526 </td><td></td></tr><tr><td > 96-подальше плоской нижней полистирола TC обработанных микропланшетов </td><td > Гранулирование </td><td > 3596 </td><td ></td></tr><tr><td > 100 мм TC обработанных блюд культуры </td><td > Гранулирование </td><td > 430167 </td><td ></td></tr><tr><td > Acrodisc шприц фильтры, 0,45 мкм </td><td > Pall Life Sciences </td><td > 4604 </td><td ></td></tr><tr><td > Агар </td><td > Teknova </td><td > A7777 </tг&gt; <td ></td></tr><tr><td > Агароза </td><td > GeneMate </td><td> E-3120-500 </td><td ></td></tr><tr><td > Ампициллин </td><td> Сигма </td><td > A0166 </td><td> 100 мг / мл в воде </td></tr><tr><td > BD FACSCanto II потока цитометр </td><td> BD Biosciences </td><td ></td><td > или аналогичный </td></tr><tr><td> BD Сокол труб круглого сечения нижней полистирола </td><td> BD Biosciences </td><td > 352054 </td><td > для FACS </td></tr><tr><td > Боск 23 клетки </td><td > АТСС </td><td > CRL-11270 </td><td ></td></tr><tr><td > CO 2 инкубатор способны 37 ° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM (среда Дульбекко изменение Орла) </td><td > Сигма </td><td> D6429 </td><td ></td></tr><tr><td > FBS (эмбриональной телячьей сыворотки) </td><td > Близнецы Био-продукты </td><td > 100-106 </td><td ></td></tr><tr><td > FITC α-крыса CD90/mouse CD90.1 антител </td><td > BioLegend </td><td > 202503 </td><td > FITCα-Thy1.1 </td></tr><tr><td > FuGENE 6 Трансфекция реагент </td><td > Roche </td><td > 11814443001 </td><td></td></tr><tr><td > HEPES буферного раствора </td><td > Invitrogen </td><td > 15630-080 </td><td ></td></tr><tr><td > KAPA HiFi ДНК-полимеразы </td><td > KAPA Biosystems </td><td > KK2101 </td><td ></td></tr><tr><td > LB бульоне (лизогении бульон - А. Р. Лурия) Порошок </td><td > Difco </td><td > 240230 </td><td ></td></tr><tr><td > МИССИЯ TRC shRNA бактериальных фондовом глицерина </td><td > Сигма </td><td ></td><td> shRNA векторов </td></tr><tr><td > NCS (новорожденного телячьей сыворотки) </td><td > Близнецы Био-продукты </td><td > 100-504 </td><td ></td></tr><tr><td> PBS (фосфатный буферный раствор) </td><td > Invitrogen </td><td> 70011 </td><td > разбавленного до 1 раза в воде </td></tr><tr><td > PE α-человеческий IgM антитела </td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS (пенициллин, стрептомицин-глютамин раствора) </td><td > Близнецы Bio-PГСМ </td><td > 400-110 </td><td ></td></tr><tr><td> Polybrene (hexadimethrine бромид) </td><td > Сигма </td><td > 107689 </td><td > 10 мг / мл в воде </td></tr><tr><td > PureYield плазмиды системы Midiprep </td><td > Promega </td><td > A2495 </td><td></td></tr><tr><td> Пуромицин </td><td> Сигма </td><td > P8833 </td><td > 250 мкг / мл в воде </td></tr><tr><td > QIAquick гель добычи комплект </td><td > QIAGEN </td><td > 28706 </td><td ></td></tr><tr><td> Рамос (РА 1) клетки </td><td > АТСС </td><td > CRL-1596 </td><td ></td></tr><tr><td > RPMI-1640 </td><td > Сигма </td><td > R8758 </td><td ></td></tr><tr><td > Надстрочный II </td><td > Invitrogen </td><td > 18064-022 </td><td></td></tr><tr><td > SYBR БЫСТРО КПЦР комплект </td><td > KAPA Biosystems </td><td > KK4601 </td><td ></td></tr><tr><td > Taq-ДНК-полимераза </td><td > Invitrogen </td><td > 18038-042 </td><td ></td></tr><tr><td > ТОПО Т. А. Клонирование комплект </td><td > Инвитропоколения </td><td > K4520-01 </td><td ></td></tr><tr><td > TRIzol </td><td > Invitrogen </td><td > 15596-026 </td><td ></td></tr><tr><td > Мастер С. В. Геномная ДНК система очистки </td><td > Promega </td><td > A2361 </td><td ></td></tr><tr><td > X-Gal [5-бром-4-хлор-3-indoyl-β-D-galatopyranoside] </td><td > Growcells </td><td > C-5687 </td><td > 40 мг / мл в ДМСО </td></tr></tbody></table>

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

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 immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 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

Мы опишем, как выполнять ретровирусных или лентивирусов инфекции гиперэкспрессия или shRNA содержащих конструкций в человеческой Рамос B-клеточной линии и как измерить соматических hypermutation в этих клетках.

Оценка соматических Hypermutation в Рамос-клеток после Сверхэкспрессия или Нокдаун специфических генов

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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Поколение дискриминационных человеческих моноклональных антител от редких антиген специфические клетки, циркулирующие в крови

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Оценка клеточного стресса и воспаления в дискретных окситоцина секретирующих ядрах головного мозга новорожденных крыс до и после первого кормления молозивом

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Анализ миграционных потока индуцированного сдвига мышиных маргинальные зоны B клеток In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...