CK是由哥伦比亚大学的研究生课程。 UB是白血病和淋巴瘤协会的美国，从白血病研究基金会的收件人的新研究者奖的研究员，是由哥伦比亚大学新进教师启动经费支持。

1。脾B淋巴细胞的分离和刺激<ol><li>援助缺陷小鼠12岁之间的2-3个月收获的脾脏。脾隔离在无菌条件下进行，并暂时保存在冷磷酸盐缓冲液（PBS）中含有15％胎牛血清（FBS），机关。 </li><li>脾，然后转移到无菌组织培养罩，和2.5％胎牛血清的PBS在5毫升匀浆。匀浆液转移到15毫升的猎鹰管。 </li><li> 1000RPM离心10分钟脾组织匀浆样品在4 ° C至颗粒细胞。 </li><li>离心后弃上清。重悬细胞沉淀在10毫升红细胞（RBC）的裂解缓冲液在室温下7分钟，以避免红血细胞的污染。 </li><li>离心机在1000 RPM为10分钟，倒出上清液样本，并重新悬浮在10毫升PBS含2.5％FBS细胞。 </li><li>取出100μL分装和1:1000稀释使用标准hemacytometer使用台盼蓝排除任何死细胞计数细胞。大约有20-30万个细胞，可每脾。 </li><li> 1000RPM样品离心10分钟，倒出上清液。 </li><li> 0.5％BSA / PBS含2mm的EDTA浓度在10月7日每90μL细胞重悬沉淀。 </li><li>加入抗CD43抗体包被的磁珠细胞特异性结合非B细胞。使用90μL，10％微升珠（10 7细胞）。拌匀，孵育4℃20分钟的彗星。 </li><li>一旦细胞都被贴上了，加入2.5％FBS / PBS顶部管冲洗。在10分钟1000转离心样品。 </li><li>倾倒上清液。在每毫升10 × 10 7细胞的浓度在0.1％的BSA / PBS重悬细胞。 </li><li>装上一个磁选机的磁排序列。下面的位置，直接列一个锥形管，收集流过负载3ML 0.5％BSA / PBS总理和洗列。 </li><li>一旦流经洗涤液，更换一个新的收集管。如果样品是小于1毫升样本量，调整至1 mL，用0.5％BSA / PBS / EDTA的负载列中的样本。负荷最大只有1毫升每列单元格，如果有必要使用多列。 </li><li>允许细胞被动流经列和列下未结合的细胞收集在锥形管。洗净用3 mL 0.5％BSA / PBS，同时继续收集流列的4倍。 </li><li>所有流量通过CD43负休息B细胞将丰富的收集。丢弃列。离心机流过，在10分钟1000转。 </li><li>排出上清液，加入10毫升培养基（15％的FCS / +谷氨酰胺，PEN /链球菌，非必需氨基酸，50微米Β- ME公司） </li><li>计数在每毫升10 6细胞的细胞和文化。 </li><li>为了刺激B细胞的生长和增殖，补充中期，终浓度20μg/ mL和1μg/ mL的重组IL - 4和抗CD40抗体，分别和细胞转移到培养瓶中。 </li><li>文化过夜的细胞。计数细胞，并稀释至10 6细胞每毫升，加入适当的IL - 4和CD40 。 </li><li>文化细胞病毒感染前72小时。 </li></ol> 2。生产者细胞的转染使用转染的既定协议，无论是使用阳离子脂质或磷酸钙。转染10厘米3板，每DNA的构造，使用100微克每盘的质粒DNA。我们经常使用磷酸钙转染法13，14，并注意与氯喹的补充时，病毒滴度高。 <ol><li>使用60％-70％汇合凤凰细胞作为生产细胞转染。在此汇合细胞计数，应每10厘米的板5万细胞。 </li><li>转染前一小时，更换凤凰细胞生长的新鲜完整的媒体与媒体。 </li><li>在无菌eppendorpf管，结合包装构造的DNA，250μL0.5M氯化钙2 100微克，并用无菌水调整最终体积为500μL </li><li>用1毫升吸管，大力500μL2X国家警察混合在15毫升的聚丙烯管这个解决方案</li><li>允许这种混合物在室温下休息8-10分钟。在这段时间内会形成沉淀。 </li><li>添加下拉明智的细胞，而纷飞的介质均匀分布的混合物整个转染混合物。 </li><li>细胞在37℃，12个小时。 </li><li>转染后12小时，更换8毫升完整的B细胞生长介质中的细胞恢复到37℃培养箱。 </li></ol> 3。刺激脾B细胞的感染<ol><li>去除上清液凤凰细胞48小时后transfection和上清液通过0.2微米的过滤器，以消除任何细胞碎片。 </li><li>混合3毫升病毒上清含3毫升预激活的B细胞（如步骤1）。为了加强病毒吸附到细胞，加入终浓度为每毫升16微克的聚凝胺。 </li><li>细胞感染了90分钟在2500转离心30 ° C。 </li><li>后立即旋转，为进一步48-72小时，在37 ° C，让细胞生长和增殖。 </li></ol> 4。流式细胞仪分析<ol><li>使用流式细胞仪分析细胞的表面表达B220和表面IgG1的。在细胞表面的IgG1的存在表明，类开关重组已经通过的援助基因的逆转录病毒互补抢救成功的结果发生。 </li></ol> 5。代表性的成果我们已经成功地救出蛋白因子活化诱导胞苷脱氨酶（AID），从援助缺陷（AID - / - ）的不足，采用逆转录病毒转导的B淋巴细胞。简而言之，我们产生了鼠标援助（MAID）蛋白表达，转染与PMX - MAID - GFP 15-18凤凰细胞重组逆转录病毒。收获病毒感染进入细胞在体外类开关重组（CSR）的刺激条件下19援助缺陷小鼠的缺陷援助乙。感染的B细胞，然后分析了CSR公司的IgG1的文化后使用流式细胞仪分析72hrs。图2显示的IgG1的检测在B细胞的援助，但不是空的构造表面，说明CSR公司作为援助表达救援的结果发生。 <img src="/files/ftp_upload/2371/2371fig1.jpg" alt="图1" /> 图1：计划内的生产者细胞的逆转录病毒包装：PMX - IRES - GFP基因的兴趣，Ψ代表，亚克隆入，如前面所述 15 。质粒转染到能够生产GAG - POL和包膜蛋白（凤凰，Orbigen），使用磷酸钙的方法13，14 ecotropic病毒包装细胞系。转染后48个小时，上清液中含有的病毒粒子感染收获到目标主乙从小鼠细胞。 <img src="/files/ftp_upload/2371/2371fig2.jpg" alt="图2" /> 图2：援助缺陷的B细胞，刺激抗CD40和IL - 4是一个包含PMX - MAID - GFP或单独表达GFP逆转录病毒的逆转录病毒感染。流式细胞仪分析，企业社会责任，IgG1的水平进行了评估。

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这里描述和图1描绘的逆转录病毒转导的脾B细胞是一种遗传性的方法，是在B淋巴细胞的研究非常有用，因为许多lymphopoesis发展的事件是由转录调控1 ，2控制。 ，在B细胞的成熟，通过CD40L的触发，后期是诱导B细胞生长，进入细胞周期， 和增殖11，20所必需的。 如图2所示，B细胞可以在体外刺激抗CD40和IL - 4，经过这一阵扩散，逆转录病毒载体的功能在B细胞?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>22400</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>20012</td>
<td>&nbsp;</td>
<tr>
<td>Red Blood Cell (RBC) lysis buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>CD43 beads</td>
<td>&nbsp;</td>
<td>Miltenyi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B Cell Complete Media</td>
<td>&nbsp;</td>
<td>Various components</td>
<td>Various Components</td>
<td>RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50&mu;M &beta;-Mercaptoethanol</td>
</tr>
<tr>
<td>IL-4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD40</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>553787</td>
<td>&nbsp;</td>
<tr>
<td>polybrene</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>107689 &#x2028;</td>
<td>&nbsp;</td>
<tr>
<td>Chloroquine diphosphate salt</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>C6628</td>
<td>Used at 100mM</td>
</tr>
<tr>
<td>Phoenix Eco cells (Murine)</td>
<td>&nbsp;</td>
<td>Orbigen</td>
<td>RVC-10002</td>
<td>&nbsp;</td>
<tr>
<td>PE-Cy5-&alpha;-mouse-CD45R (B220)</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>15-0452-81</td>
<td>&nbsp;</td>
<tr>
<td>PE-&alpha;-mouse-IgG1</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>A85-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

recombinant retroviral production and infection of b cells

在真核细胞基因的转基因表达是一个功能强大的反向遗传学方法，在其中一个感兴趣的基因是异源表达系统的控制下表达，以方便所产生的表型分析。这种方法可以用来在生物体中发现一个不正常的基因表达，表达了一个基因产物的突变形式，或过度表达的基因产物的显性负形式。它是造血系统的研究，转录调控是一个主要控制机制，在发展和分化的B 细胞在2-4审查，特别有用。 小鼠遗传学是研究人类基因和疾病的有力工具。一个小鼠和人类基因组的比较分析，揭示了共线性的保护，在超过90％的基因组5此外，在小鼠模型所使用的技术是适用于人类基因的研究，例如， 基因 干扰和等位基因更换6。然而，转基因小鼠的建立需要一个很大的金融和技术性质资源。有几个项目已经开始编译库（KOMP，EUCOMM，NorCOMM）敲除小鼠品系或突变引起的菌株（理研），这需要大规模的努力和合作 7的。因此，这是可取的，首先研究的一个发展的小鼠模型前，在原代细胞的细胞培养模型所需的基因表型。 逆转录病毒的DNA整合到宿主DNA中，最好内或附近转录单位或CpG岛，稳定和遗传利益打包基因的表达，同时避免转录沉默 8 9。然后下一个高效率的逆转录病毒启动子控制转录的基因，在转录和蛋白质的生产效率高。因此，逆转录病毒表达可用于难转染的细胞，细胞在有丝分裂过程中的一个活跃的状态。由于该病毒的结构基因内包装细胞系中，用于克隆感兴趣的基因的表达载体含有病毒没有结构基因，这既消除了病毒的回复的可能性，并增加了安全与病毒上清工作如没有传染性的病毒粒子产生的 10 。 在这里，我们提出了一个脾B细胞重组逆转录病毒的生产和后续感染的协议。隔离后，刺激培养的脾细胞与钍派生淋巴因子和抗CD40，诱导B细胞的增殖和分化 11爆裂。此协议是为年底发生在B细胞发育和分化，B细胞是从以下初始造血事件，但在抗原刺激诱导浆细胞分化的脾孤立的事件研究的理想。

Watch this Scientific Journal Video about Recombinant Retroviral Production and Infection of B Cells at JoVE.com

Recombinant Retroviral Production and Infection of B Cells

一个有效率的制度中的一个基因的结构和功能分析<em&gt;体外</em&gt;脾B淋巴细胞的文化描述。这种方法需要利用一个辅助的自由，ecotrophic包装细胞系的重组逆转录病毒的生产。稳定，遗传兴趣在初级淋巴细胞基因的表达，是实现领导在接受类开关重组的B细胞表面抗体的生成。

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

recombinant-retroviral-production-and-infection-of-b-cells

Research

JoVE Journal

Immunology and Infection

14.0K 次观看.  Columbia University College of Physicians and Surgeons. 一个有效率的制度中的一个基因的结构和功能分析体外脾B淋巴细胞的文化描述。这种方法需要利用一个辅助的自由，ecotrophic包装细胞系的重组逆转录病毒的生产。稳定，遗传兴趣在初级淋巴细胞基因的表达，是实现领导在接受类开关重组的B细胞表面抗体的生成。

视频: Recombinant Retroviral Production and Infection of B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

科研

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

重组细小病毒与腺病毒派生系统的帮助下高效生产

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

乙型肝炎病毒感染原代肝细胞和库珀细胞的 "肝片上" 培养

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...