Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jul 1;88(13):5611–5615. doi: 10.1073/pnas.88.13.5611

Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system.

P Piccioli 1, F Ruberti 1, S Biocca 1, A Di Luzio 1, T M Werge 1, A Bradbury 1, A Cattaneo 1
PMCID: PMC51927  PMID: 1712102

Abstract

We present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles.

Full text

PDF
5611

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Biocca S., Neuberger M. S., Cattaneo A. Expression and targeting of intracellular antibodies in mammalian cells. EMBO J. 1990 Jan;9(1):101–108. doi: 10.1002/j.1460-2075.1990.tb08085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Byrne G. W., Ruddle F. H. Multiplex gene regulation: a two-tiered approach to transgene regulation in transgenic mice. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5473–5477. doi: 10.1073/pnas.86.14.5473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Capecchi M. R. The new mouse genetics: altering the genome by gene targeting. Trends Genet. 1989 Mar;5(3):70–76. doi: 10.1016/0168-9525(89)90029-2. [DOI] [PubMed] [Google Scholar]
  4. Cattaneo A., Neuberger M. S. Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain. EMBO J. 1987 Sep;6(9):2753–2758. doi: 10.1002/j.1460-2075.1987.tb02569.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cuello A. C., Galfre G., Milstein C. Detection of substance P in the central nervous system by a monoclonal antibody. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3532–3536. doi: 10.1073/pnas.76.7.3532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cuello A. C. Peptides as neuromodulators in primary sensory neurons. Neuropharmacology. 1987 Jul;26(7B):971–979. doi: 10.1016/0028-3908(87)90075-x. [DOI] [PubMed] [Google Scholar]
  7. Engvall E., Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep;8(9):871–874. doi: 10.1016/0019-2791(71)90454-x. [DOI] [PubMed] [Google Scholar]
  8. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gamse R., Holzer P., Lembeck F. Decrease of substance P in primary afferent neurones and impairment of neurogenic plasma extravasation by capsaicin. Br J Pharmacol. 1980 Feb;68(2):207–213. doi: 10.1111/j.1476-5381.1980.tb10409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  11. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  12. Holzer P., Gamse R., Lembeck F. Distribution of substance P in the rat gastrointestinal tract--lack of effect of capsaicin pretreatment. Eur J Pharmacol. 1980 Feb 8;61(3):303–307. doi: 10.1016/0014-2999(80)90132-6. [DOI] [PubMed] [Google Scholar]
  13. Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
  14. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  15. Lee V. M., Page C. The dynamics of nerve growth factor-induced neurofilament and vimentin filament expression and organization in PC12 cells. J Neurosci. 1984 Jul;4(7):1705–1714. doi: 10.1523/JNEUROSCI.04-07-01705.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levi-Montalcini R., Angeletti P. U. Second symposium on catecholamines. Modification of sympathetic function. Immunosympathectomy. Pharmacol Rev. 1966 Mar;18(1):619–628. [PubMed] [Google Scholar]
  17. Levi A., Eldridge J. D., Paterson B. M. Molecular cloning of a gene sequence regulated by nerve growth factor. Science. 1985 Jul 26;229(4711):393–395. doi: 10.1126/science.3839317. [DOI] [PubMed] [Google Scholar]
  18. Lewis S. A., Cowan N. J. Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum. Mol Cell Biol. 1986 May;6(5):1529–1534. doi: 10.1128/mcb.6.5.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mayo K. E., Warren R., Palmiter R. D. The mouse metallothionein-I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell. 1982 May;29(1):99–108. doi: 10.1016/0092-8674(82)90094-0. [DOI] [PubMed] [Google Scholar]
  20. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  21. Nilsson J., von Euler A. M., Dalsgaard C. J. Stimulation of connective tissue cell growth by substance P and substance K. Nature. 1985 May 2;315(6014):61–63. doi: 10.1038/315061a0. [DOI] [PubMed] [Google Scholar]
  22. Orlandi R., Güssow D. H., Jones P. T., Winter G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989 May;86(10):3833–3837. doi: 10.1073/pnas.86.10.3833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pernow B. Substance P. Pharmacol Rev. 1983 Jun;35(2):85–141. [PubMed] [Google Scholar]
  24. Rossant J. Manipulating the mouse genome: implications for neurobiology. Neuron. 1990 Mar;4(3):323–334. doi: 10.1016/0896-6273(90)90045-h. [DOI] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ward E. S., Güssow D., Griffiths A. D., Jones P. T., Winter G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature. 1989 Oct 12;341(6242):544–546. doi: 10.1038/341544a0. [DOI] [PubMed] [Google Scholar]
  28. Werge T. M., Biocca S., Cattaneo A. Intracellular immunization. Cloning and intracellular expression of a monoclonal antibody to the p21ras protein. FEBS Lett. 1990 Nov 12;274(1-2):193–198. doi: 10.1016/0014-5793(90)81362-r. [DOI] [PubMed] [Google Scholar]
  29. van den Pol A. N., Decavel C., Levi A., Paterson B. Hypothalamic expression of a novel gene product, VGF: immunocytochemical analysis. J Neurosci. 1989 Dec;9(12):4122–4137. doi: 10.1523/JNEUROSCI.09-12-04122.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES