Name: 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
Uploaded: 2020-05-31T13:00:00
Description: Watch this Scientific Journal Video about 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 at JoVE.c

这项工作得到了 1R01 HL107490、1R01 HL136098、P01 HL055798 项目 3、P01 HL136275-01（C.A. McNamara）和 R01GM100776（T.P. Bender）的支持。A. Upadhye 由美国心脏协会博士后奖学金 16PRE3030002 和 5T32AI007496-20 提供支持。我们感谢弗吉尼亚大学流细胞测量核心的乔安妮·兰尼根、迈克·索尔加和克劳德·丘的出色技术援助。

所有动物协议都得到弗吉尼亚大学动物护理和使用委员会的批准。
1. 圆锥形B-1细胞的磁分离和浓缩
<ol><li>使用 CO2对 12-14 周大、男性、CD45.1+ApoE-/-鼠标进行安乐死。</li>
	<li>使用笔直的手术剪刀在腹部进行表面切割，并用弯曲的剪刀剥去皮肤，露出腹壁。使用 10 mL 注射器和 25 G 针，以 10 mL 37 °C RPMI-1640 介质的 10 mL 冲洗穿孔腔。摇动鼠标以分离细胞，?...

<ol>
	<li>Baumgarth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+Cell+Heterogeneity+and+the+Regulation+of+Natural+and+Antigen-Induced+IgM+Production.">B-1 Cell Heterogeneity and the Regulation of Natural and Antigen-Induced IgM Production.</a> Frontiers in Immunology. 7, 324 (2016).</li><li>Holodick, N. E., Vizconde, T., Rothstein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1a+cell+diversity:+nontemplated+addition+in+B-1a+cell+Ig+is+determined+by+progenitor+population+and+developmental+location.">B-1a cell diversity: nontemplated addition in B-1a cell Ig is determined by progenitor population and developmental location.</a> Journal of Immunology. 192 (5), 2432-2441 (2014).</li><li>Prohaska, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+Parallel+Sequencing+of+Peritoneal+and+Splenic+B+Cell+Repertoires+Highlights+Unique+Properties+of+B-1+Cell+Antibodies.">Massively Parallel Sequencing of Peritoneal and Splenic B Cell Repertoires Highlights Unique Properties of B-1 Cell Antibodies.</a> Journal of Immunology. 200 (5), 1702-1717 (2018).</li><li>Upadhye, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversification+and+CXCR4-Dependent+Establishment+of+the+Bone+Marrow+B-1a+Cell+Pool+Governs+Atheroprotective+IgM+Production+Linked+to+Human+Coronary+Atherosclerosis.">Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis.</a> Circulation Research. 125 (10), 55-70 (2019).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mechanisms+define+murine+B+cell+lineage+immunoglobulin+heavy+chain+(IgH)+repertoires.">Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires.</a> Elife. 4, 09083 (2015).</li><li>Choi, Y. S., Dieter, J. A., Rothaeusler, K., Luo, Z., Baumgarth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+cells+in+the+bone+marrow+are+a+significant+source+of+natural+IgM.">B-1 cells in the bone marrow are a significant source of natural IgM.</a> European Journal of immunology. 42 (1), 120-129 (2012).</li><li>Holodick, N. E., Tumang, J. R., Rothstein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+secretion+by+B1+cells:+Differential+intensity+and+IRF4-dependence+of+spontaneous+IgM+secretion+by+peritoneal+and+splenic+B1+cells.">Immunoglobulin secretion by B1 cells: Differential intensity and IRF4-dependence of spontaneous IgM secretion by peritoneal and splenic B1 cells.</a> European Journal of Immunology. 40 (11), 3007-3016 (2010).</li><li>Moon, H., Lee, J. G., Shin, S. H., Kim, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS-induced+migration+of+peritoneal+B-1+cells+is+associated+with+upregulation+of+CXCR4+and+increased+migratory+sensitivity+to+CXCL12.">LPS-induced migration of peritoneal B-1 cells is associated with upregulation of CXCR4 and increased migratory sensitivity to CXCL12.</a> Journal of Korean Medical Science. 27 (1), 27-35 (2012).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+plasma+cell+alloantigen+1+defines+layered+development+of+B-1a+B-cell+subsets+with+distinct+innate-like+functions.">Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (49), 20077-20082 (2012).</li><li>Yang, Y., Tung, J. W., Ghosn, E. E., Herzenberg, L. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+and+differentiation+of+natural+antibody-producing+cells+in+mouse+spleen.">Division and differentiation of natural antibody-producing cells in mouse spleen.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (11), 4542-4546 (2007).</li><li>Tsiantoulas, D., Gruber, S., Binder, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+cell+immunoglobulin+directed+against+oxidation-specific+epitopes.">B-1 cell immunoglobulin directed against oxidation-specific epitopes.</a> Frontiers in Immunology. 3, 415 (2012).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines:+Valuable+tools+or+useless+artifacts.">Cell lines: Valuable tools or useless artifacts.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>McMahon, S. B., Norvell, A., Levine, K. J., Monroe, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+of+murine+B+lymphocyte+blasts+as+a+method+for+examining+gene+regulation+in+primary+B+cells.">Transient transfection of murine B lymphocyte blasts as a method for examining gene regulation in primary B cells.</a> Journal of Immunological Methods. 179 (2), 251-259 (1995).</li><li>Lin, K. I., Calame, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+genes+into+primary+murine+splenic+B+cells+using+retrovirus+vectors.">Introduction of genes into primary murine splenic B cells using retrovirus vectors.</a> Methods in Molecular Biology. 271, 139-148 (2004).</li><li>Moghimi, B., Zolotukhin, I., Sack, B. K., Herzog, R. W., Cao, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Efficiency+Ex+Vivo+Gene+Transfer+to+Primary+Murine+B+Cells+Using+Plasmid+or+Viral+Vectors.">High Efficiency Ex Vivo Gene Transfer to Primary Murine B Cells Using Plasmid or Viral Vectors.</a> Journal of Genetic Syndromes and Gene Therapy. 2 (103), 1000103 (2011).</li><li>DeKoter, R. P., Lee, H. J., Singh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PU.1+regulates+expression+of+the+interleukin-7+receptor+in+lymphoid+progenitors.">PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors.</a> Immunity. 16 (2), 297-309 (2002).</li><li>Holodick, N. E., Vizconde, T., Hopkins, T. J., Rothstein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Decline+in+Natural+IgM+Function:+Diversification+and+Selection+of+the+B-1a+Cell+Pool+with+Age.">Age-Related Decline in Natural IgM Function: Diversification and Selection of the B-1a Cell Pool with Age.</a> The Journal of Immunology. 196 (10), 4348-4357 (2016).</li><li>Laurie, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+and+efficient+expression+in+mouse+and+human+B+cells+by+a+novel+hybrid+immunoglobulin+promoter+in+a+lentiviral+vector.">Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector.</a> Gene Therapy. 14 (23), 1623-1631 (2007).</li><li>Warncke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vitro+transduction+of+naive+murine+B+cells+with+lentiviral+vectors.">Efficient in vitro transduction of naive murine B cells with lentiviral vectors.</a> Biochemical and Biophysical Research Communications. 318 (3), 673-679 (2004).</li><li>Moon, B. G., Takaki, S., Miyake, K., Takatsu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+IL-5+for+mature+B-1+cells+in+homeostatic+proliferation,+cell+survival,+and+Ig+production.">The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production.</a> Journal of Immunology. 172 (10), 6020-6029 (2004).</li><li>Takatsu, K., Kouro, T., Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+5+in+the+link+between+the+innate+and+acquired+immune+response.">Interleukin 5 in the link between the innate and acquired immune response.</a> Advances in Immunology. 101, 191-236 (2009).</li><li>Baumgarth, 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+double+life+of+a+B-1+cell:+self-reactivity+selects+for+protective+effector+functions.">The double life of a B-1 cell: self-reactivity selects for protective effector functions.</a> Nature Reviews Immunology. 11 (1), 34-46 (2011).</li></ol>

此处提供的方法可实现稳定且相对高效的原发B-1a细胞基因传递、体内采用转移以及注射细胞的识别和定位。细胞在细胞转移后17周被检测到，并保留增加的CXCR4表达。逆转录病毒介导的传递使原鼠B-1a细胞的转导效率为30-40%，对我们手中的细胞生存能力的影响最小（图4e）。这与Moghimi及其同事先前的一项研究的结果一致，该研究将基因转移技术与原发性鼠B细胞（包括逆转录病...

最近的研究表明，相当多的免疫细胞，特别是B细胞，皮质和功能异质性取决于细胞定位11，2，3，4，5。2,3,4,5B-1a细胞是具有异质性产生保护性IgM抗体的杂质能力的一个群体;骨髓B-1a细胞分泌IgM，对血浆IgM成形10剂6有显著贡献，而心内性B-1a细胞在平衡性时具有低水平IgM分泌，而是可以通过先天多收费的受体（TLR）或细胞因子介导的信号激活，以迅速增殖、迁移和分泌IgM7、8、9。7,8,9,B-1a细胞IgM抗体可识别病原体、凋亡细胞和氧化LDL上存在的氧化特异性表皮（OSE），与OSE结合的IgM可以预防动脉粥样硬化11等疾病的炎症性下游信号。因此，通过增加对骨髓等部位的细胞迁移来增加IgM产量的策略可能具有治疗性。然而，这种策略必须针对和细胞类型特定，因为非目标效应可能对免疫功能或健康产生负面影响。
在这里，我们描述了一种在原鼠B-1a细胞中针对和长期过度表达CXCR4的方法，以及随后的采用转移，以可视化细胞迁移和功能IgM抗体的产生（图1）。与转化细胞系的转染相比，原发B细胞的基因操作受到转染效率低的限制。然而，由于转化的细胞系可以显著偏离原细胞1212，13，13使用原细胞可能会提供的结果，更紧密地配合正常生理。在原鼠B细胞的基因转移中，已经描述了几种技术，包括逆转录病毒转导、脱病毒转导、脂肪化或电穿孔型转染，其效率、转化性和对细胞健康的影响13、14、15。,1513,以下方法采用逆转录病毒转导，因为它产生了足够的基因转移效率>30%，同时对细胞生存能力的影响最小。CXCR4表达逆转录病毒是使用前面描述的逆转录病毒构造鼠干细胞-内部核糖体进入部位-绿色荧光蛋白（MSCV-IRES-GFP） 产生的;MigR1）16，其中小鼠CXCR4基因被子克隆4。MigR1（控制（Ctl）-GFP和CXCR4-GFP逆转录病毒颗粒是使用磷酸盐钙转染产生的，如先前公布的协议44、1414所述。
成功转导的B-1a细胞随后被静脉注射转移到淋巴细胞缺乏的Rag1-/----小鼠中。供体小鼠和受体小鼠还含有对脂蛋白E（ApoE）基因的敲除，导致OSE积累和动脉粥样硬化增加，从而为体内B-1细胞活化和IgM生产提供了一个模型。此外，供体小鼠和受体小鼠在CD45等位型上有所不同;供体B-1细胞来自CD45.1+ApoE-/-----小鼠，并转移到Rag1-/- CD45.2+ApoE-/------受体。这允许在移植后将供体CD45.1与受体CD45.2 B细胞区分，而无需在流细胞测量分析期间为B细胞标记额外染色。这里提供的结果表明，B-1a细胞上的目标CXCR4过度表达与B-1a细胞迁移到骨髓的能力增强有关，骨髓与血浆抗OSE IgM增加有关。我们还提供了一种通过负选择来丰富圆锥体B-1细胞的方法，并演示了B-1细胞激活对有效转导的要求。该方法可应用于其他逆转录病毒构造，以研究蛋白质过度表达对B-1a细胞迁移、表型或功能的影响。此外，使用CD45.1与CD45.2等位基因区分理论上可以允许转移到含有内源性B细胞的其他免疫足够的鼠型。

<table>
	<tbody>
		<tr>
			<td>70 micron filter caps</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-biotin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD16/CD32, or Fc block</td>
			<td>Life Technologies</td>
			<td>MFCR00</td>
			<td></td>
		</tr>
		<tr>
			<td>B220 APC</td>
			<td>eBioscience</td>
			<td>17-0452-83</td>
			<td>Clone: RA3-6B2</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19 APCef780</td>
			<td>eBioscience</td>
			<td>47-0193-82</td>
			<td>Clone: eBio1D3</td>
		</tr>
		<tr>
			<td>CD23 biotin</td>
			<td>eBioscience</td>
			<td>13-0232-81</td>
			<td>Clone: B3B4</td>
		</tr>
		<tr>
			<td>CD23 PECy7</td>
			<td>eBioscience</td>
			<td>25-0232-82</td>
			<td>Clone: B3B4</td>
		</tr>
		<tr>
			<td>CD3e biotin</td>
			<td>eBioscience</td>
			<td>13-0033-85</td>
			<td>Clone: eBio500A2</td>
		</tr>
		<tr>
			<td>CD45.1 ApoE-/- mice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in house</td>
		</tr>
		<tr>
			<td>CD45.1 PerCP-Cy5.5</td>
			<td>BD Biosciences</td>
			<td>560580</td>
			<td>Clone: A20</td>
		</tr>
		<tr>
			<td>CD45.2 BV421</td>
			<td>BD Biosciences</td>
			<td>562895</td>
			<td>Clone: 104</td>
		</tr>
		<tr>
			<td>CD45.2 Rag1-/- ApoE-/- mice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in house</td>
		</tr>
		<tr>
			<td>CD5 PE</td>
			<td>eBioscience</td>
			<td>12-0051-83</td>
			<td>Clone: 53-7.3</td>
		</tr>
		<tr>
			<td>Ctl-GFP retrovirus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Generated in house using GFP-expressing retroviral plasmid MigR1 provided by Dr. T.P. Bender</td>
		</tr>
		<tr>
			<td>CXCR4 APC</td>
			<td>eBioscience</td>
			<td>17-9991-82</td>
			<td>Clone: 2B11</td>
		</tr>
		<tr>
			<td>CXCR4-GFP retrovirus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Generated in house by cloning mouse CXCR4 into MigR1 retroviral plasmid</td>
		</tr>
		<tr>
			<td>F4/80 biotin</td>
			<td>Life Technologies</td>
			<td>MF48015</td>
			<td>Clone: BM8</td>
		</tr>
		<tr>
			<td>Flowjo Software v. 9.9.6</td>
			<td>Treestar Inc.</td>
			<td>License required</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr-1 biotin</td>
			<td>eBioscience</td>
			<td>13-5931-82</td>
			<td>Clone: RB6-8C5</td>
		</tr>
		<tr>
			<td>heat-inactivated fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM PECF594</td>
			<td>BD Biosciences</td>
			<td>562565</td>
			<td>Clone: R6-60.2</td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BD Biosciences</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>029405</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Yellow</td>
			<td>Life Technologies</td>
			<td>L34968</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>NK1.1 biotin</td>
			<td>BD Biosciences</td>
			<td>553163</td>
			<td>Clone: PK136</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>ODN 1668</td>
			<td>InvivoGen</td>
			<td>tlrl-1668</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119 biotin</td>
			<td>eBioscience</td>
			<td>13-5921-82</td>
			<td>Clone: Ter119</td>
		</tr>
	</tbody>
</table>

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

由于细胞功能受细胞微环境中利基特定因素的影响，剖析细胞定位和迁移的方法可以进一步洞察细胞功能。B-1a细胞是小鼠中一个独特的B细胞子集，在健康和疾病期间产生保护性天然IgM抗体，防止氧化特异性表皮。B-1a细胞IgM的产生因B-1a细胞位置而异，因此从治疗的角度来看，将B-1a定位目标定位到支持高抗体生产的利基领域变得有用。在这里，我们描述了一种方法，通过逆转录病毒介导的C-X-C图案化学酶受体4（CXCR4）的过度表达，将B-1a细胞迁移到骨髓。原鼠B细胞的基因诱导可能具有挑战性，通常根据技术产生10-20%的低转染效率。在这里，我们证明原鼠B-1a细胞的逆转录病毒转导可产生30-40%的转染效率。该方法利用转导B-1a细胞的采用细胞转移到B细胞缺陷受体小鼠中，使供体B-1a细胞迁移和定位得到可视化。该协议可用于其他逆转录病毒结构，可用于收养后转移后的各种功能检测，包括对供体细胞或宿主细胞表型和功能的分析，或B-1a细胞转移后分泌的可溶性因子的分析。使用以CD45.1和CD45.2等位类型区分的不同供体和受体小鼠，并在逆转录病毒质粒体内存在GFP报告器，还可以检测含有内源性B细胞群的其他免疫充足的小鼠模型中的供体细胞。

图 1给出了协议的概述。图 2显示了其他锥形细胞类型的磁耗竭后，圆锥形 B-1a 细胞的富集。与F4/80+ 巨噬细胞相比，后耗尽分数中的活单细胞的CD19+B细胞比例+更高，缺乏CD5HI CD19- T细胞，并且与预耗尽分数相比，CD19+ CD5中B-1a细胞的频率增加。图 3显示了 B 细胞激活对成功逆转录病毒 B 细胞?...

Watch this Scientific Journal Video about 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 at JoVE.c

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

在这里，我们描述了一种抗逆转录病毒过度表达和采用迁移的鼠B-1a细胞的方法，以检查体内B-1a细胞的迁移和定位。该协议可扩展到不同的下游功能检测，包括定量供体B-1a细胞定位或分析供体细胞衍生分泌因子的收养后转移。

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 ...

该协议可以评估靶向基因传递如何影响B-1a细胞的定位和功能，并可以作为研究体内基因治疗潜力的概念验证方法。这种方法为原发性B-1a细胞提供了稳定且相对有效的基因传递，并允许识别供体细胞表型和功能后收养转移。该方法可用于检查体内不同的供体和宿主细胞过程，包括细胞存活、增殖和功能，并可应用于其他采用性转移系统。永久液体的收获可能比较棘手。一定要彻底脱离细胞，以最大限度地提高恢复，并避免刺穿肠道，可以污染你的性细胞群体。对于腹膜B-1细胞的收集，使用直手术剪刀在腹部做一个表面切割的12至14周大，男性，CD45.1+阿波利波蛋白E敲除小鼠，并使用弯曲的剪刀剥回皮肤，露出腹膜壁。使用配备 25 量针的 10 毫升注射器，用 10 毫升 37 摄氏度 RPMI-1640 介质冲洗腹腔。并抓住尾巴的底座，让鼠标从一侧完全摇动到另一侧15至20秒。摇动后，使用注射器从臀部水平正上方的右下侧吸出液体，靠近肠道，注意避免破坏表皮脂肪库和基础器官。收集六到七毫升的熔岩后，将液体放入冰上50毫升的锥形管中。使用钳子抓住隔膜上方的腹膜壁，使动物垂直升高，使任何剩余的液体留在腹膜腔的底部。使用手术剪刀在肝脏上方的内皮内壁上做一个小切口，而不切割肝脏本身。并使用玻璃移液器和灯泡收集任何剩余的内层液体。收集完所有液体后，将所有小鼠的内酮洗净细胞等为一体，并在50毫升的冰管中，将每管浓度的10至第八细胞添加1倍至第八个细胞。通过离心沉淀细胞。将每粒颗粒重新用一毫升的抗CD16/CD32抗体在检测缓冲液中以1比50的比例稀释，在4摄氏度下孵育10分钟。在孵育结束时，将同等体积的生物素化抗体主混合添加到每个管中，在4摄氏度下孵育20分钟，然后在每管5毫升的测定缓冲液中清洗。根据细胞浓度和制造商的建议，用适当体积的抗生物素微珠和适当的孵化持续时间重新填充颗粒，然后每管洗5毫升测定缓冲液。将每个颗粒重新悬浮在 500 微升的测定缓冲液中，并用每柱三毫升的测定缓冲液填充适当数量的磁性选择柱。将细胞转移到底向柱上，在15毫升的圆锥管中收集含有丰富B-1细胞的埃卢特。然后用额外的测定缓冲液清洗每个磁性选择柱，直到总收集的体积为10毫升，并在B细胞培养基中每毫升浓度的10倍将净化后细胞分数重新悬浮到第六细胞。刺激内胸乙基细胞至少留出1倍10到第七细胞进行非转导控制，将剩余的细胞分成两个相等的体积进行转导。稀释细胞至1.5倍至5细胞每150微升的中等浓度，并添加150微升细胞到一个96孔圆底板的井中，以进行转导。然后在每孔中加入100个TLR9激动剂纳米摩尔，并将板放在细胞培养培养箱中16至18小时。用于逆转性转导的内粒B细胞解冻磷酸钙转染逆转录病毒颗粒物在冰上，并立即添加到控制和化疗受体逆转录病毒超级杀菌剂到适当的板的每个井和多重感染20至1在存在8微克每毫升多聚苯乙烯和新鲜的β-汞醇在55微摩尔最终浓度。当所有病毒被添加后，通过离心使细胞旋转，并在细胞培养箱中放置板三个小时。在孵育结束时，收获细胞，在新鲜的B细胞培养基中重新育制，并隔夜孵育。与其他内膜细胞类型的磁耗后，与F4/80+巨噬细胞相比，消耗后部分的活单细胞具有更大的CD19+B细胞比例，并且缺乏CD5hi CD19-T细胞，并且它们含有比消耗前分数更高的CD19+CD5中等B-1a细胞的频率。成功转导的GGFP+B-2、B-1、B-1a和B-1b细胞子集的频率增加与感染病毒多样性的增加直接相关。与使用 24 孔或 6 孔板相比，使用 96 孔圆底板可进一步提高转导效率。CXCR4-GFP逆转录病毒转导可诱导成功的B-1细胞CXCR4过度表达，并增加体外向CXCL12的迁移，而不会对B细胞的生存能力产生重大影响。此外，在细胞转移后17周从CD45.2受接受小鼠的骨髓和脾殖中恢复后，采用移植CD45.1+供体细胞维持其CXCR4过度表达。值得注意的是，CXCR4表达和供体细胞定位到骨髓而不是脾脏之间的正关联已经确定。骨髓中供体细胞数与抗MDA低密度脂蛋白IgM的血浆量之间观察到正关联。许多额外的技术可用于回答诸如目标基因传递对疾病发展的影响，或者哪些捐赠者获得影响宿主生理学的分泌因素。

Research

JoVE Journal

Immunology and Infection

Recombinant Retroviral Production and Infection of B Cells

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 ...

科研

免疫与感染

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...