Histogram of the Cerulean reporter expressed in J558L cells infected with or without viruses (a) and A20, Bal17 and M12 cells infected with viruses (b). delivery system, others and Aniracetam we have demonstrated that this mouse IgE BCR exhibits elevated activity compared to the IgG1 BCR in the absence of cognate antigen [2, 3]. Using the same approach to express chimeric B cell receptors in main B cells, we have Aniracetam characterized the contribution of different domains of the IgE BCR to this antigen-independent activity [2]. Compared to main B cells, immortalized B cell lines offer some unique advantages to study BCR signaling. For example, unlike main B cells, some B cell lines do not Colec11 need the tonic transmission activity of the BCR for survival. As a result, it is possible to reconstitute BCR signaling in B cell lines by adding or removing specific components in these cells. Additionally, B cell lines can also be managed for extended periods and can be cultured in large quantities, making them suitable for biochemical studies. Exogenous genes are usually delivered to B cell lines by chemical transfection and electroporation. Numerous B cell lines, such WEHI-231, BAL17, and M12 cells, can also be readily transduced by retroviruses [4]. We have analyzed the cell surface translocation of IgE, IgG1, and their chimeric receptors delivered to J558L cells by retroviral vectors [2]. In most cases, the recombinant retroviruses used to transduce main B cells and B cell lines are replication incompetent. To generate such recombinant retroviruses, the gene of interest is cloned into a plasmid-based retroviral vector in place of the viral genes. The and genes encode nucleocapsid (Gag) and envelope (Env) viral proteins respectively, while encodes protease, reverse transcriptase, and integrase. These viral proteins are necessary for retrovirus packaging and replication. To render the recombinant computer virus replication incompetent, the viral genes are either supplied in a separate plasmid vector and/or stably integrated in a packaging cell collection [5]. Once a B cell is usually infected with recombinant retrovirus, the vector DNA with the exogenous gene will integrate into the genome of the B cell, resulting in the stable maintenance and expression of the exogenous gene in the B cell. Detailed protocols, from your construction of a retroviral vector, to the contamination of murine main B cells and B cell lines, are explained below. 2.?Materials 2.1. Construction of Retroviral Vector Cloning vectors, such as pCR2.1 (Invitrogen). Retroviral vectors, such as pQEF-T2A-Cerulean, pQCXIN (Clontech), MSCV-IRES-GFP (Addgene). Packing pladmids: pCL-Eco (Addgene) or MSCV ecotopic gag-pol-env plasmid (G/P/E). Molecular biology reagents, such as restriction enzymes, ligase, qualified cells, plasmid DNA preparation kit. 2.2. Preparation of Recombinant Retrovirus, In Vitro Culture of Main B Cells, and Spinfection of the Cultured Main B Cells with Retrovirus Total DMEM medium (cDMEM): DMEM high glucose medium with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 penicillin/streptomycin/L-glutamine. Opti-MEM reduced serum medium (Invitrogen). (((gene of interest, long terminal repeat, 3 self-inactivating LTR, extended packaging Aniracetam transmission. EF-1: human EF-1 promoter. AmpR, ampicillin resistance gene cassette Using standard molecular biology techniques, genes of interest can be cloned into the pQEF-Ceru-T2A vector following the procedures explained below and layed out in Fig. 1. A similar strategy could be used to clone genes of interest into other retroviral vectors, such as MSCV-IRES-GFP. Clone desired expression cassette into a common cloning vector, such as pCR2.1. Verify the expression cassette by sequencing. Clone the sequence-verified expression cassette from your cloning vector into the retroviral vector. For cloning into the pQEF-Ceru-T2A retroviral vector, the expression cassette is usually released from your cloning vector by digestion with restriction enzymes SnaBI and XhoI. Gel purify the expression cassette, and ligate with gel-purified pQEF-Ceru-T2A that has been digested with the same restriction enzymes. Transform qualified cells with the ligation sample. Prepare plasmid DNA of the putative recombinant retroviral vector. Verify the identity of the clones by restriction enzyme digestion. 3.2. Preparation of Recombinant Retrovirus, In Vitro Culture of Main B Cells, and Spinfection of the Cultured Main B Cells with Retrovirus We always use Aniracetam freshly generated retroviruses to infect mouse B cells. The whole experiment, including preparing recombinant retroviruses, setting up the B cell culture, infecting cultured B cells with the retroviruses, and analyzing the transduced B cells, takes about 6 Aniracetam days. Some of these parts of the experiment are overlapping. For better arranging and executing the experiment, we find it.