Difference between revisions of "Part:BBa K1391100:Experience"

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Experiment 1:
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  Localization of receptor to the cell membrane
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In our first experiment, we aimed to determine if the engineered B-cell receptor components (CD79A, CD79B, IgM Heavy Chain, and Kappa Light Chain) were able to assemble to form a receptor complex and then localize to the cell membrane. Since the beta-amyloid oligomers characteristic of Alzheimer's disease accumulate in the extracellular matrix of the brain, it is important that the receptor membrane localize so that it can detect these plaques outside the cell.
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To determine the localization of the receptors, we immunostained using IgM specific antibodies. We analyzed the immunostained samples in two ways. The first was through flow cytometry analysis. This method enabled us to determine whether the antibodies bound to the outside of our cells, which would indicate that the B-cell receptor's IgM component had reached the membrane. We also used confocal microscopy to visualize the localization of our receptor inside our cells by permeabilizing the cells and incubating them with anti-IgM antibodies.
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For samples that were analyzed using flow cytometry, we transiently transfected HEK293 cells with plasmids encoding constitutive expression (hEF1a promoter) of the engineered B-cell receptor components along with hEF1a:mKate2 (constitutive red fluorescent protein) as a transfection marker. The transfection marker provides an indication of approximately how many plasmids are uptaken by a particular cell, which helps to connect plasmid number to observed output levels. We then treated cells with anti-IgM antibodies conjugated to Alexa Fluor 488 (yellow fluorescent dye). By measuring yellow output relative to red output using the flow cytometer, we hoped to be able to compare plasmid number to anti-IgM antibody binding, where high levels of red fluorescence (many plasmids) would correspond to high levels of yellow fluorescence (high levels of antibody binding, meaning a high level of BCR surface expression).

Revision as of 15:49, 20 October 2014


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Applications of BBa_K1391100

B-cell receptors (BCRs) are multiprotein immune receptors found exclusively on the surface of B cells. The BCR multiprotein complex is centered around a membrane-bound IgM antibody formed from two light chains and two heavy chains. This antibody is bound to two accessory proteins CD79A and CD79B. When the antibody binds to an extracellular antigen, receptors dimerize resulting in the phosphorylation of the intracellular tails of CD79A and CD79B by the tyrosine-protein kinase Lyn. In response, another cofactor, spleen tyrosine kinase (Syk), is recruited to the receptor and phosphorylated, initiating a signalling cascade that results in the proliferation of the activated B cells. This receptor is important in clonal selection of B cells during human immune response.

This particular sequence is the heavy chain for an antibody against beta amyloid meant to be used in a B-Cell Receptor. It is meant to be used in conjunction with an associated light chain as well as CD79A, CD79B, Lyn, and Syk.

User Reviews

UNIQf07773586050f851-partinfo-00000000-QINU UNIQf07773586050f851-partinfo-00000001-QINU Experiment 1: 

 Localization of receptor to the cell membrane

In our first experiment, we aimed to determine if the engineered B-cell receptor components (CD79A, CD79B, IgM Heavy Chain, and Kappa Light Chain) were able to assemble to form a receptor complex and then localize to the cell membrane. Since the beta-amyloid oligomers characteristic of Alzheimer's disease accumulate in the extracellular matrix of the brain, it is important that the receptor membrane localize so that it can detect these plaques outside the cell.

To determine the localization of the receptors, we immunostained using IgM specific antibodies. We analyzed the immunostained samples in two ways. The first was through flow cytometry analysis. This method enabled us to determine whether the antibodies bound to the outside of our cells, which would indicate that the B-cell receptor's IgM component had reached the membrane. We also used confocal microscopy to visualize the localization of our receptor inside our cells by permeabilizing the cells and incubating them with anti-IgM antibodies.

For samples that were analyzed using flow cytometry, we transiently transfected HEK293 cells with plasmids encoding constitutive expression (hEF1a promoter) of the engineered B-cell receptor components along with hEF1a:mKate2 (constitutive red fluorescent protein) as a transfection marker. The transfection marker provides an indication of approximately how many plasmids are uptaken by a particular cell, which helps to connect plasmid number to observed output levels. We then treated cells with anti-IgM antibodies conjugated to Alexa Fluor 488 (yellow fluorescent dye). By measuring yellow output relative to red output using the flow cytometer, we hoped to be able to compare plasmid number to anti-IgM antibody binding, where high levels of red fluorescence (many plasmids) would correspond to high levels of yellow fluorescence (high levels of antibody binding, meaning a high level of BCR surface expression).