Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-04-04
2000-06-13
Campell, Bruce R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 721, 435366, 4353723, 424 9321, C12N 500, G01N 3353, A01N 6300
Patent
active
060748362
DESCRIPTION:
BRIEF SUMMARY
The invention is related to a method of marking eukaryotic (mammalian) cells by the use of a cell surface receptor with a modified intracellular domain as a selectable marker.
The identification of cells into which a DNA sequence has been successfully introduced is an essential step in recombinant DNA technology. As the DNA sequence to be introduced will not necessarily lead to a phenotype which may be selected in a simple manner, there is usually introduced an additional DNA sequence coding for a selectable phenotype. There is still only a very limited number of such selectable marker genes for use in recombinant DNA technology of eukaryotic cells. Most of these available marker genes, such as, for example, the Herpes simplex virus type I thymidine kinase (Wigler et al., Cell 1 (1977) 223) or hypoxanthine phosphoribosyl transferase (Jolly et al., Proc. Nat. Acad. Sci. 80 (1983) 477) correspond to genes, or are highly related to genes, which are constitutively expressed in the majority of normal cells or are identical with such genes.
Therefore, these marker genes can only be used in special mutant cells which do not express the corresponding gene. A preferred marker gene on the other hand should not be expressed in the majority of mammalian cells or at least not be expressed in certain tissues and cell types, so that they can be used as selectable marker genes in recombinant DNA technology involving these cells.
Pawlink et al. have described the use of the CD24 cell surface antigen as a dominant selectable marker in retroviral mediated gene transfer (Journal of Cellular Biochemistry, Supplement 17 E, page 203, abstract S210). With this marker NIH-3T3 fibroblasts, BAF-3 pre-B cells as well as murine bone marrow cells could be labelled by retroviral infection and the labelled cells were detected by fluorescence activated cell sorting analysis. However, as the CD24 cell surface antigen is normally expressed on some mammalian cells, the use of this marker will be limited by the difficulty to differentiate between cells normally expressing the CD24 cell surface antigen and cells labelled with this marker.
Furthermore, it has been found that the CD24 cell surface antigen, following the heterologous expression, is presented at the cell surface to a little extent only.
It was therefore the object of the invention to provide a process for marking eukaryotic cells, with the use of cell surface receptors, by means of which process the cell surface receptors are presented to a large extent at the cell surface, whereby the sensitivity of the selection and of the detection of marked cells can be increased.
This object is accomplished by a process of marking a eukaryotic (mammalian) cell by expressing in this cell a nucleic acid, the nucleic acid encoding a cell surface receptor, and by presenting the receptor at the cell surface, the process being characterized by the use of a nucleic acid in which the region encoding the intracellular domain of the receptor is completely or partly deleted, or modified so that the receptor presented at the surface carilot effect any signal transduction after binding to its binding partner.
Preferably, the intracellular domain is completely deleted, or individual nucleotides are modified.
Preferably, there is used the nucleic acid encoding the NGF-, CD24- or the LDL receptor.
The sequence of the human NGF receptor, which will be referred to hereinafter as "low affinity nerve growth factor receptor, LNGFR", is described in D. Johnson, Cell 47 (1986) 545-554. Preferably, there is used a nucleic acid in which the DNA region encoding the amino acids 245 (beginning with nucleotide 930) to the C-terminus has been deleted (cf. SEQ ID NO:1).
In SEQ ID NO:1 the extracellular domain is coded by the nucleotides 114 (ATG)--863, the transmembrane domain is coded by the nucleotides 864-929, and the intracellular domain is coded by the nucleotides 930-1394 (Reddy et al., Molecular Brain Research 8 (1990) 137-141)
For the deletion of the intracellular domain it is possible to delete the complete domain
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Bordignon Claudio
Mavilio Fulvio
Boehringer Mannheim GmbH
Campell Bruce R.
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