Stillman, C. A., Linton, P. J., Koutz, P. J., Decker, D. J., Klinman, N. R., Gingeras, T. R. (1994) Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification. PCR Methods and Applications, 3 (6). pp. 320-331. ISSN 10549803 (ISSN) (Public Dataset)
Abstract
The isothermal 3SR amplification method has been employed to assist in cloning the V(L) and V(H) genes from cells of hybridomas and splenic fragment cultures expressing antibodies for phosphorylcholine (PC) and estradiol (E2), respectively. As a first step, pools of degenerate primer pairs were identified complementary to immunoglobulin light and heavy chain variable (V) genes and capable of amplifying immunoglobulin RNA specifically at 42°C. To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC V(H) and V(L) cDNAs were joined together to form a single chain (sc) antibody construct and were expressed in Escherichia coli under the regulation of the alkaline phosphatase (phoA) promoter. Similarly, the combination of a murine spleen fragment and 3SR methodologies were employed to clone a selected pool of cDNAs for cultures producing anti-estradiol antibodies. This approach of using the murine spleen fragment and 3SR isothermal amplification offers the advantages of B-cell follicle architecture for antigen-driven B-cell maturation and proliferation and RNA-specific amplification, respectively. The potential utility of these advantages for the production of monoclonal antibodies and for providing the capability of studying memory B-cell development are discussed.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | complementary dna immunoglobulin immunoglobulin heavy chain immunoglobulin light chain phosphorylcholine article cell line escherichia coli hybridoma immunoglobulin gene immunoglobulin variable region molecular cloning mouse nonhuman priority journal Amino Acid Sequence Animal Antibodies, Monoclonal Base Sequence Cloning, Molecular DNA Replication Estradiol Hybridomas Immunoglobulins Mice Mice, Inbred BALB C Molecular Sequence Data Polymerase Chain Reaction Spleen Tissue Culture |
| Subjects: | Investigative techniques and equipment > cloning Investigative techniques and equipment > assays > cloning bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > monoclonal antibody |
| CSHL Authors: | |
| Communities: | CSHL labs > Gingeras lab |
| Depositing User: | CSHL Librarian |
| Date: | 1994 |
| Date Deposited: | 13 Mar 2012 15:39 |
| Last Modified: | 23 Jan 2015 14:33 |
| Related URLs: | |
| Dataset ID: | |
| URI: | https://repository.cshl.edu/id/eprint/25258 |
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