This work was supported by 1R01 HL107490, 1R01 HL136098, Project 3 of P01 HL055798, P01 HL136275-01 (C.A. McNamara), and R01GM100776 (T.P. Bender). A. Upadhye was supported by American Heart Association Pre-doctoral fellowship 16PRE30300002 and 5T32AI007496-20. We thank Joanne Lannigan, Mike Solga, and Claude Chew from the University of Virginia Flow Cytometry Core for their excellent technical assistance.

All animal protocols were approved by the Animal Care and Use Committee at the University of Virginia.
1. Magnetic separation and enrichment of peritoneal B-1 cells
<ol>
	<li>Euthanize a 12&#8722;14-week-old, male, CD45.1+ApoE-/- mouse using CO2.</li>
	<li>Make a superficial cut in the abdomen using straight surgical scissors and peel back skin using curved scissors to expose the peritoneal wall. Flush peritoneal cavity with 10 mL of 37 &#176;C RPMI-1640 medium using a 10 mL syringe and 25 G needle. Shake mouse to disengage cells by grasping the tail and moving the mouse side to side thoroughly for 15&#8722;20 s. 
	NOTE: Massaging the lavaged peritoneum can also maximize cell recovery.</li>
	<li>Collect peritoneal fluid using a 10 mL syringe and 25 G needle by drawing up fluid at the lower right side of the peritoneum just above the level of the hip, near the intestines. Avoid disrupting epidydimal fat depots and underlying organs. Avoid drawing fluid from the mouse&#8217;s left side as the omental fat can easily be drawn into the syringe.</li>
	<li>Once ~6&#8722;7 mL of fluid is collected, dispense into a 50 mL conical tube placed on ice. Next, elevate the mouse vertically by holding the peritoneal wall above the diaphragm using forceps so that any remaining fluid remains at the bottom of the peritoneal cavity. Make a small cut in the peritoneal wall using surgical scissors above the liver (make sure not to cut the liver) and collect any remaining peritoneal fluid using a glass pipet and bulb.</li>
	<li>Pool peritoneal washout cells from all CD45.1+ApoE-/- mice (n = 15&#8722;20) into 50 mL conical tubes, and store on ice. 
	NOTE: For calculating the number of mice needed: B-1a cells comprise 5&#8722;10% of the total peritoneal population and yield roughly 2.5&#8722;5 x 105 B-1a cells per mouse in authors&#8217; hands. Transduction efficiency is ~30&#8722;40%, as shown below.</li>
	<li>Count live cells using a viability dye such as trypan blue and a hemocytometer (dilute samples 1:5 in trypan blue and load 10 &#181;L into the hemocytometer chamber). Pool cells at up to 1 x 108 cells per tube. Centrifuge cells at 400 x g for 5 min at 4 &#176;C, then aspirate supernatant.</li>
	<li>Resuspend up to 1 x 108 cells in 1 mL of anti-CD16/CD32 antibody (Table of Materials) diluted 1:50 in assay buffer (1x phosphate buffered saline [PBS], 0.5% bovine serum albumin [BSA], 2 mM EDTA) in order to block Fc receptors. Scale as necessary based on cell count. Incubate on ice for 10 min at 4 &#176;C.</li>
	<li>Prepare a 2x master mix of the biotinylated antibodies (Table 1) in assay buffer for depletion. The 2x master mix accounts for the volume of the liquid the cells are already in when incubating with anti-CD16/CD32. For example, if cells are incubating in 500 &#181;L of diluted anti-CD16/CD32, then add 500 &#181;L of 2x master mix containing 10 &#181;L of biotinylated Ter119, Gr-1, CD23, and NK1.1 antibodies, and 25 &#181;L of biotinylated F4/80 antibody to achieve the final concentrations given in Table 1. Add 2x master mix to cells and stain for 20 min at 4 &#176;C.</li>
	<li>Wash cells with 5 mL of assay buffer and centrifuge at 400 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Resuspend and incubate cells with anti-biotin microbeads (Table of Materials) diluted in assay buffer as per the concentration and protocol recommended by the manufacturer.</li>
	<li>Wash with 5 mL of assay buffer and centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate supernatant and resuspend up to 1 x 108 cells in 500 &#181;L of assay buffer.</li>
	<li>Prime magnetic selection columns (Table of Materials) with 3 mL of assay buffer. Transfer cells onto primed magnetic selection columns and collect the eluent containing enriched B-1 cells in a 15 mL conical tube on ice. Wash the magnetic selection column with additional assay buffer till the overall volume collected is 10 mL. 
	NOTE: An aliquot of the pre-purified and post-purified cell fractions should be separately analyzed by flow cytometry for purification efficiency by staining for CD19+ B cells, F480+ macrophages, and CD5+ T cells as shown in Figure 2.</li>
	<li>Count the post-purified cell fraction (the eluent containing enriched B-1 cells) by counting live cells as in step 1.6, and resuspend at 1 x 106 cells/mL in B cell culture medium (RPMI-1640, 10% heat-inactivated fetal bovine serum [FBS], 10 mM HEPES, 1x non-essential amino acids, 1 mM sodium pyruvate, 50 &#181;g/mL gentamicin, 55 &#181;M &#946;-mercaptoethanol).</li>
</ol>
2. Peritoneal B-1 cell stimulation
<ol>
	<li>Split pooled cells for the two transduced conditions (Ctl-GFP and CXCR4-GFP), while setting aside and plating at least 10 x 106 cells for a non-transduced control.</li>
	<li>Plate up to 150 &#181;L (150,000 cells) per well into 96-well round-bottom plates.</li>
	<li>Add 100 nM TLR9 agonist ODN1668 to all wells to stimulate cell proliferation.</li>
	<li>Incubate for 16&#8722;18 h at 37 &#176;C, 5% CO2.</li>
</ol>
3. Retroviral transduction of peritoneal B cells
<ol>
	<li>Generate Ctl-GFP (MigR1) and CXCR4-GFP retroviral particles using calcium phosphate transfection as described in previously published protocols14. 
	NOTE: This will take several days, so prepare and titer retroviral stocks and store aliquots at -80 &#176;C prior to starting this protocol.</li>
	<li>Thaw retrovirus stocks on ice. Use immediately and do not re-freeze as virus titer significantly diminishes during freeze-thaw cycles. Add Ctl-GFP or CXCR4-GFP retroviral supernatants at 20:1 multiplicity of infection (MOI) to cells, in the presence of 8 &#181;g/mL polybrene and fresh &#946;-mercaptoethanol at 55 &#181;M final concentration. Do not add viral stocks to the cells set aside for the non-transduced control.</li>
	<li>Perform spinfection by centrifuging plates at 800 x g for 90 min at room temperature.</li>
	<li>Incubate plates with retrovirus at 37 &#176;C, 5% CO2 for an additional 3 h.</li>
	<li>Harvest and replate cells in fresh B cell medium and incubate at 37 &#176;C, 5% CO2 overnight.</li>
</ol>
4. Cell sorting of transduced peritoneal B-1a cells
<ol>
	<li>Harvest cultured cells and pool into three separate 50 mL conical tubes for each condition: non-transduced, Ctl-GFP transduced, and CXCR4-GFP transduced.</li>
	<li>Count live cells as in step 1.6, then centrifuge at 400 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Resuspend cells at 100,000 cells/&#181;L in sort buffer (PBS + 1% BSA) containing 1:50 anti-CD16/CD32 antibody (Table of Materials) in order to block Fc receptors. Incubate on ice for 10 min at 4 &#176;C.</li>
	<li>Aliquot cells for compensation controls (~30,000 cells per compensation control): for unstained and single stain controls aliquot from non-transduced sample and for GFP single stain control aliquot from transduced samples. 
	NOTE: Commercially available compensation beads can alternatively be used for single stain controls if non-transduced cell number is low.</li>
	<li>Prepare a 2x antibody master mix containing the fluorophore-conjugated antibodies in sort buffer (Table 1). Add 2x master mix to non-transduced, CTL-GFP transduced, and CXCR4-GFP transduced samples. Add individual antibodies to single stain controls. Incubate for 20 min at 4 &#176;C in the dark. 
	NOTE: The 2x master mix accounts for the volume of liquid the cells are already in when incubating with anti-CD16/CD32, as in step 1.8. An aliquot of cells from each condition can separately be stained for CXCR4 to confirm CXCR4 overexpression.</li>
	<li>Wash samples with 1 mL of sort buffer and strain through 70 &#181;m filters into polypropylene tubes.</li>
	<li>Centrifuge at 400 x g for 5 min at 4 &#176;C and aspirate supernatant. Resuspend samples at 50,000 cells/&#181;L in sort buffer.</li>
	<li>Prior to cell sorting prepare labeled fluorescence-activated cell sorting (FACS) collection tubes containing 1 mL of collection medium (RPMI-1640, 20% heat-inactivated FBS, 10 mM HEPES, 1x non-essential amino acids, 1 mM sodium pyruvate, 50 &#181;g/mL gentamicin, 55 &#181;M &#946;-mercaptoethanol) for each population to be sorted.</li>
	<li>Prior to running samples on the cell sorter add 2x DAPI (prepared as 1:5000 dilution in sort buffer) for dead cell discrimination.</li>
	<li>Sort GFP+ B-1a cells into FACS tubes containing collection medium as DAPI- CD19+ GFP+ B220mid-lo CD23- IgM+ CD5+ cells from the CTL-GFP and CXCR4-GFP samples. Use the non-transduced sample to set the GFP+ gate and to sort DAPI- CD19+ GFP- B220mid-lo CD23- IgM+ CD5+ non-transduced B-1a cells. 
	NOTE: Alternatively, non-transduced cells can also be sorted from the CTL-GFP and CXCR4-GFP samples by separately gating B-1a cells within the GFP- fraction. Transduced or non-transduced B-1b cells can also be sorted using this sort strategy as DAPI- CD19+ GFP+/- B220mid-lo CD23- IgM+ CD5- cells.</li>
</ol>
5. Adoptive transfer
<ol>
	<li>After cell sorting, centrifuge cells at 400 x g for 5 min at 4 &#176;C and aspirate supernatant carefully.</li>
	<li>Resuspend cells in cold sterile 1x PBS at 1,000 cells/&#181;L.</li>
	<li>Anesthetize male Rag1-/- ApoE-/- mice using isoflurane and inject 100 &#181;L (100,000 cells) per mouse via intravenous retro-orbital or tail vein injection using an ultra-fine insulin syringe. Inject a few mice with 1x PBS as a control.</li>
</ol>
6. Quantification of donor cells and plasma IgM
<ol>
	<li>At the desired time post-adoptive transfer, analyze transferred cells in recipient mice by bone marrow and spleen tissue harvest4 and flow cytometry. Quantify donor cell localization and CXCR4 overexpression by staining for CD45.1, CD45.2, and CXCR4 and analyze flow cytometry results using a software such as Flowjo. 
	NOTE: See Table 1 for antibodies used in this step. Ensure not to use an antibody conjugate that fluoresces in the FITC channel as GFP will be present on transduced donor cells.</li>
	<li>Isolate plasma by adding 10 &#181;L of 0.5 M EDTA to whole blood collected via cardiac puncture at the time of animal sacrifice. Centrifuge whole blood at 7,000 x g and aliquot plasma into separate 1.5 mL centrifuge tubes. Store at -80 &#176;C until performing analysis of secreted factors such as IgM by ELISA4.</li>
</ol>

<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>

The authors have nothing to disclose.

The method provided here enables stable and relatively efficient primary B-1a cell gene delivery, in vivo adoptive transfer, and identification and localization of injected cells. Cells were able to be detected 17 weeks post-cell transfer and retained increased CXCR4 expression. Retrovirus-mediated delivery yielded 30-40% transduction efficiency of primary murine B-1a cells with minimal impact on cell viability in our hands (Figure 4e). This is in line with results from a previous study by M...

Recent studies have demonstrated considerable immune cell, and specifically B cell, phenotypic and functional heterogeneity depending on cell localization1,2,3,4,5. B-1a cells are one such population with heterogeneous capacity to produce protective IgM antibodies; bone marrow B-1a cells secrete IgM constitutively and contribute significantly to plasma IgM titers6, while peritoneal B-1a cells have low-level IgM secretion at homeostasis and instead can be activated through innate toll-like receptor (TLR) or cytokine-mediated signaling to rapidly proliferate, migrate, and secrete IgM7,8,9,10. B-1a cell IgM antibodies recognize oxidation-specific epitopes (OSE) that are present on pathogens, apoptotic cells, and oxidized LDL, and IgM binding to OSE can prevent inflammatory downstream signaling in diseases like atherosclerosis11. Therefore, strategies to increase IgM production via increasing peritoneal B-1a cell migration to sites like the bone marrow may be therapeutically useful. However, it is important for such strategies to be targeted and cell-type specific, as off-target effects may negatively impact immune function or health.
Here we describe a method for targeted and long-term overexpression of CXCR4 in primary murine B-1a cells and subsequent adoptive transfer to visualize cell migration and functional IgM antibody production (Figure 1). Genetic manipulation of primary B cells is limited by low transfection efficiencies compared to transfection of transformed cell lines. However, as transformed cell lines can significantly deviate from primary cells12,13, the use of primary cells is likely to provide results that more closely align to normal physiology. Several techniques have been described for gene transfer in primary murine B cells, including retroviral transduction, adenoviral transduction, lipofection, or electroporation-based transfection, which have varying levels of efficiency, transience, and impact on cell health13,14,15. The following method utilized retroviral transduction as it yielded adequate gene transfer efficiency of &#62;30% while minimally impacting cell viability. The CXCR4-expressing retrovirus was generated using the previously described retroviral construct murine stem cell virus-internal ribosomal entry site-green fluorescent protein (MSCV-IRES-GFP; MigR1)16, into which the mouse CXCR4 gene was sub-cloned4. MigR1 (control(Ctl)-GFP) and CXCR4-GFP retroviral particles were generated using calcium phosphate transfection as described in previously published protocols4,14.
Successfully transduced B-1a cells were then intravenously transferred into lymphocyte-deficient Rag1-/- mice. Both donor and recipient mice additionally contained knockout of the apolipoprotein E (ApoE) gene, which results in increased OSE accumulation and atherosclerosis, thereby providing a model for in vivo B-1 cell activation and IgM production. Moreover, donor and recipient mice differed in CD45 allotype; donor B-1 cells came from CD45.1+ ApoE-/- mice and were transferred into Rag1-/- CD45.2+ ApoE-/- recipients. This allowed differentiation of donor CD45.1 from recipient CD45.2 B cells post-transfer without the need to additionally stain for B cell markers during flow cytometry analysis. The results provided here demonstrate that targeted CXCR4 overexpression on B-1a cells associates with increased ability of B-1a cells to migrate to the bone marrow, which associates with increased plasma anti-OSE IgM. We additionally provide a method for the enrichment of peritoneal B-1 cells through negative selection and demonstrate the requirement of B-1 cell activation for efficient transduction. This method can be adapted for other retroviral constructs to study the effect of protein overexpression on B-1a cell migration, phenotype, or function. Moreover, the use of CD45.1 versus CD45.2 allotype distinction could theoretically allow transfer into other immune-sufficient murine models containing endogenous B cells.

<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

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.

An overview of the protocol is given in Figure 1. Figure 2 displays enrichment of peritoneal B-1a cells after magnetic depletion of other peritoneal cell types. Live singlet cells in the post-depletion fraction have a greater proportion of CD19+ B cells compared to F4/80+ macrophages, lack CD5hi CD19- T cells, and contain an increased frequency of CD19+ CD5mid B-1a cells compared to the pre-depletion fractio...

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

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.

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

retroviral-overexpression-cxcr4-on-murine-b-1a-cells-adoptive

Research

JoVE Journal

Immunology and Infection

5.0K Views.  University of Virginia. 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.

Video: 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

Recombinant Retroviral Production and Infection of B Cells

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 of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

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 ...

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

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

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

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

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

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

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

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

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

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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

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.

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

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

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

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

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