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 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.
Retroviral transduction of splenic B cells as described here and as depicted in Figure 1 is a genetic approach that is useful in the study of B-lymphocytes because many of the developmental events in lymphopoesis are controlled by transcriptional regulation 1, 2. In the later stages of B cell maturation, triggering via CD40L is essential for the induction of B cell growth, entry into the cell cycle, and proliferation 11, 20. As shown in Figure 2, B cells can be stim…
The authors have nothing to disclose.
C.K. is supported by Columbia University Graduate program. U.B. is Fellow of the Leukemia and Lymphoma Society of America, the recipient of New Investigator Award from the Leukemia Research Foundation and is supported by Columbia University New Faculty start-up funds.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
FCS | Atlanta Biologicals | S11550 | ||
RPMI | Invitrogen/Gibco | 22400 | ||
PBS | Invitrogen/Gibco | 20012 | ||
Red Blood Cell (RBC) lysis buffer | Sigma Aldrich | R7757 | ||
CD43 beads | Miltenyi | |||
B Cell Complete Media | Various components | Various Components | RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50μM β-Mercaptoethanol | |
IL-4 | ||||
Anti-CD40 | BD Pharmigen | 553787 | ||
polybrene | Sigma Aldrich | 107689 | ||
Chloroquine diphosphate salt | Sigma Aldrich | C6628 | Used at 100mM | |
Phoenix Eco cells (Murine) | Orbigen | RVC-10002 | ||
PE-Cy5-α-mouse-CD45R (B220) | eBioscience | 15-0452-81 | ||
PE-α-mouse-IgG1 | BD Pharmigen | A85-1 |