Sequence Analyses from Single
Antigen-Specific B Lymphocytes

The UNC Lineberger Comprehensive Cancer Center is studying B lymphocyte development, activation and biology, with emphasis on the biology of B-1 cells and the events occurring early in B cell differentiation. RNAture’s GenePlate enabled the generation of four PCR products and sequences from a single cell to determine how antibody specificity is encoded.
The Lineberger Comprehensive Cancer Center at the University of North Carolina in Chapel Hill, the publicly assisted comprehensive cancer center for the state of North Carolina, attempts to reduce cancer occurrence and death through research, treatment, training and outreach. It is one of 40 National Cancer Institute (NCI)-designated Comprehensive Cancer Centers and one of eight Specialized Programs of Research Excellence (SPORE) in Breast Cancer in the country.
The research in the laboratory focuses on B lymphocyte development, activation and biology. In particular, they are striving to understand the biology of B-1 cells – the population of
B cells that give rise to most human and mouse B cell lymphomas. They are also focusing on the events that occur early in B cell differentiation. They hypothesize that, like T cells, B cells undergo a required positive selection event based on the specificity of the heavy chain variable region of the pre-B cell receptor. This event may have profound consequences for the shaping of the B cell receptor repertoire.
The human immune system distinguishes self from nonself using cell-mediated and humoral immune responses. Antibodies, or immunoglobulins, are the key elements to the humoral immune response. It is an immune response (chiefly against bacterial invasion) mediated through a body fluid, usually by B cells.

The hypervariable residues of both the heavy and light chains on the immunoglobulins form the antigen-binding site and determine specificity. During B cell differentiation, the immunoglobulin heavy (VH) and light (VL) chain genes undergo rearrangement to increase antibody diversity. Here, the variable regions were sequenced and analysed from a single antigen-specific B lymphocyte in order to determine the heavy-light chain pairing used to encode the specificity. Lymphocytes develop from stem cells that originate early in fetal life, reside in bone marrow in the adult and then migrate to other lymph organs where they multiply and reside. Based on their functional characteristics, lymphocytes are classified into T cells and
B cells, distinguishable by immunocytochemical means. When stimulated,
B cells differentiate into plasma cells that produce specific antibodies against an antigen. Antibodies circulate in the plasma or are present in secretions.

Methods
Splenic B lymphocytes from VH12 Tg mice [1] were stained with anti-B220-R-PE antibody and FITC (encapsulated)-liposomes. The latter contains phosphatidyl choline (PtC) as an integral part of the liposome membrane. B220+/liposome+ cells were identified using a MoFlo flow cytometer (DakoCytomation, Fort Collins, CO). Single cells were sorted by the MoFlo into the wells of a GenePlate. In some cases, the plates were cut into 8-well strips and in others the entire plate was used. Each well contained 10 無 of Lysis Buffer (RNAture). Sorted cells were either processed immediately or frozen in Lysis Buffer (immediately frozen on dry ice and then stored at -70蚓 for 2 months or more). The MoFlo was modified to provide an undeflected trajectory for the sorted cells while deflecting the waste/unwanted stream. This is critical for the accurate positioning of cells onto the small target provided by the 10 無 of Lysis Buffer in the well. The lysis/hybridization step was performed for 90 minutes at room temperature, allowing the poly(A)+ RNA in the sample to hybridize to the immobilized oligo(dT) on the GenePlate. The well was then washed three times with 100 無 of Wash Buffer (RNAture). After the last wash, the GenePlate was centrifuged briefly to collect the remaining fluid in the bottom of the wells and all traces of Wash Buffer were removed by aspiration. The reverse transcription (RT) reaction was performed in 20 無 containing 1 x RT buffer, 0.5 mM of each dNTP, 10 mM DTT and 50 units Superscript II (Invitrogen) for 40 minutes at 42蚓. The RT step was primed via the immobilized oligo(dT), and thus the resulting cDNA was permanently bound to the plate. Initially, 1 無 of cloned ribonuclease inhibitor (Invitrogen) was included, but in more recent experiments the ribonuclease inhibitor was not used without any apparent difference. Following the RT reaction, the reverse transcriptase was inactivated by heating to 70蚓 for 10 minutes. The well was then washed three times with 100 無 of Wash Buffer. After the last wash, the GenePlate was briefly centrifuged to collect the remaining Wash Buffer, and then all liquid was removed by aspiration. The first round of PCR was performed in 25 無 or 50 無 volumes and contained 1 x PCR buffer, 1.5 mM MgCl2, 0.1 mM each dNTP, 20 痢 each primer and 1.5 units of Taq polymerase (Invitrogen). The second round of PCR was performed in 50 無 using 2 無 of the previous PCR product and contained 1 x PCR buffer, 1.5 mM MgCl2, 0.2 mM each dNTP, 80 痢 of each primer and 1.5 units of Taq polymerase. The 3’ primer was nested for Vk, VH, HGPRT and actin. Both PCR reactions were heated to 94蚓 for 3 min followed by 35 cycles of amplification (94蚓 for 3 min, oligo-dependent temperature for 40 sec, 70蚓 for 45 sec) with a final step of 70蚓 for 7 min. PCR-amplified products for Vk, VH, HGPRT and actin (Figure 1) were analyzed by gel electrophoresis. Following a PCR clean-up step, 30-40 mg of PCR product and 10 pmol primer were mixed (to 20 無 with water) and submitted to a sequencing facility (Figure 2).

Results and conclusions
Previously, the methods for obtaining PCR products used in sequencing variable regions, were generated from conventional RT-PCR reactions in 200 無 PCR tubes. Although theoretically possible, sequencing heavy and light chains from a single cell was never attempted due to tedious and time-consuming methodology. In the system described here, heavy and light chain variable region genes, as well as actin and HGPRT, from single B lymphocytes expressing a surface immunoglobulin with specificity for PtC, were PCR-amplified and sequenced (Figures 1 and 2). The use of the GenePlate easily permitted the generation of four PCR products and sequences from a single cell and can easily be scaled up, and automated if desired, for a much higher throughput then previously possible. The system described above enabled them to perform one month’s worth of research (using traditional methods) in one day.
GenePlate allows high throughput poly(A)+ RNA isolation from cells, tissue culture and suspension cells for drug screening and functional genomics applications without the need for prior total RNA purification. No filtration or centrifugation step is required for cell culture or tissue samples. Researchers can synthesize cDNA directly in the well using the immobilized oligo-(dT)20 as a primer. Because the technology enables poly(A)+ RNA selection in one simple step, researchers can obtain a more defined sample than with total RNA, but without the inconvenience and added processing time of traditional poly(A)+ RNA enrichment procedures. The purified mRNA can then be used directly for the synthesis of cDNA, amplified and quantitated by RT-PCR or recovered by elution.
GenePlate minimizes sample manipulations, resulting in lowered risk of sample contamination and less variance (lower CV’s). A coefficient of variation of less that 6% between well-to-well and plate-to-plate ensures reproducible and reliable data. The well’s oligonucleotide immobilization technology allows mRNA isolation, cDNA synthesis and PCR in the same well.
Gene expression can be detected from a single cell using RT-PCR. The well is thermal cycler compatible and functions as the solid support for RT-PCR and other applications, allowing researchers to move directly to the amplification step if desired. The simplicity of the GenePlate enables the entire process to be fully automated on virtually any automated liquid handler.

References
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