Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-06-09
2001-09-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S468000, C435S477000
Reexamination Certificate
active
06284541
ABSTRACT:
DESCRIPTION
The invention concerns a method for introducing a foreign DNA into the genome of a target cell by homologous recombination as well as suitable DNA constructs for the homologous recombination.
Methods for introducing foreign DNA into the genome of eukaryotic cells by homologous recombination are known (e.g. WO 90/11354, WO 91/09955). In this process a starting cell is transfected with a DNA construct which contains at least one and preferably two DNA sequence sections that are homologous to regions of the genome of the cell to be transfected, a positive selection marker gene and optionally a negative selection marker gene. In addition the DNA construct can contain a heterologous expression control sequence if it is intended to activate a gene which is normally silent in the transfected cell. The transfected cells are cultured under conditions in which a selection for the presence of the positive selection marker gene takes place which, on expression, leads to a selectable phenotype.
A second selection step is usually carried out in order to distinguish between cells in which a homologous recombination has taken place and cells in which the vector has only been randomly integrated into the genome of the host cell. For this a negative selection marker gene is used such as the HSV thymidine kinase gene (HSV-TK) which, when present, leads to the destruction of cells in the presence of a selection agent e.g. ganciclovir. In homologous recombination the cell loses the HSV thymidine kinase gene so that cells are resistant to ganciclovir. Cells in the genome of which the targeting vector has been incorporated by random, non-homologous integration do not lose the HSV-TK gene and are therefore sensitive towards ganciclovir. Cells are preferably used for this type of selection by HSV-TK/ganciclovir which contain no functional thymidine kinase gene (e.g. CEM tk
−
from Ogden Bioservices Corp., Rockville Md., USA, Cat. No. 491).
However, other host cells used for homologous recombination possess their own thymidine kinase gene. But this cellular thymidine kinase gene causes background problems in the negative selection. Thus for example homologously recombined clones may be lost during screening. Similar problems also occur with other negative selection marker genes which code for a gene product whose expression must be selected against after transfection.
The use of polypeptides located on the cell surface as positive transfection markers is known. Thus for example WO 95/06723 describes a method for labelling cells using a partially deleted cell surface receptor gene.
In order to avoid the problems which occur with the previously used negative selection marker genes, a negative selection marker gene is used according to the invention which codes for a polypeptide located on the cell surface.
Hence the present invention concerns a method for introducing foreign DNA into a host cell by homologous recombination in which the host cell is transfected with a recombinant vector comprising two flanking nucleotide sequences which are homologous to a target sequence in the genome of the host cell and inside of which a nucleotide sequence coding for a positive selection marker is located, and a nucleotide sequence outside the flanking sequences which codes for a negative selection marker, each of the nucleotide sequences coding for the positive and the negative selection marker being operatively linked to an expression control sequence which is active in the host cell, wherein at least one nucleotide sequence coding for a polypeptide located on the cell surface is used as the negative selection marker gene so that after integration of the DNA construct into the genome of the cell by homologous recombination the negative selection marker gene is not expressed and after a random integration of the vector into the genome of the cell the negative selection marker gene is expressed and its gene product is presented on the cell surface.
Hence according to the invention a negative selection marker gene coding for a polypeptide located on the surface is used for the homologous recombination at an appropriate site in the vector to avoid using a negative selection method with a selection agent that is toxic for the cell. A negative selection marker gene is preferably used which codes for a polypeptide which does not normally occur in the host cell.
Problems with toxicity or with background signals that have been described for TK selection do not occur in the method according to the invention. A further advantage of the method according to the invention is that the number of transfected cells that have to be examined for expression of the target gene is considerably reduced.
The host cell is preferably a eukaryotic cell, particularly preferably a mammalian cell and most preferably a human cell.
In order to identify and isolate cells in which a homologous recombination has taken place, a selection step is carried out according to the invention for the presence of the positive selection marker gene and a further selection step is carried out for the absence of the negative selection marker gene.
The selection step for the presence of the positive selection marker gene can be carried out in a conventional manner. Any selection marker gene, and especially those suitable for eukaryotic cells, whose expression results in a selectable phenotype e.g. antibiotic resistance or auxotrophy can be used as the positive selection marker gene. Antibiotic resistance genes are preferably used e.g. the neomycin, kanamycin, geneticin or hygromycin resistance gene. A particularly preferred positive selection marker gene is the neomycin phosphotransferase gene.
The negative selection marker gene used for the method according to the invention codes for a gene product which is presented on the surface of the host cell, preferably for a membrane-based polypeptide. Preferred examples of such membrane-based polypeptides are the LNGF, the CD24, the LDL or the trk receptor or a membrane-based receptor fragment containing the ligand binding domain of the respective receptor. Suitable receptor fragments in which the intracellular domain is completely or partially deleted or is modified in such a manner that the receptor presented on the surface cannot cause signal transduction are described in WO 95/06723. A particularly preferred example of such a receptor fragment is a deletion mutant of the LNGF receptor (dLNGFR) which is a fragment of the human low-affinity receptor of the nerve growth factor whose intracellular and signal transducing domains have been deleted (WO 95/06723).
The principle of homologous recombination under negative selection by dLNGFR is shown schematically in FIG.
1
. This selection principle can of course be applied to other selection marker genes coding for surface-associated polypeptides. A plasmid is used as the recombinant vector which contains two flanking nucleic acid sections (HR1, HR2) homologous to the desired target sequence and between them the positive selection marker gene, the neomycin resistance gene (NeoR). A nucleotide sequence coding for dLNGFR is arranged on the plasmid outside the two flanking homologous nucleotide sequences.
The regions HR1, NeoR and HR2 are integrated into the genome when a homologous recombination occurs with a region in the area of the target gene (HR). However, the sequence coding for dLNGFR is not integrated into the genome. In contrast the dLNGFR gene is retained in a form capable of expression when the plasmid is randomly integrated into the genome of the host cell.
The selection according to the invention for the absence of the negative selection marker gene in the transfected host cell preferably comprises the steps:
(a) contacting the transfected cell with a binding molecule which binds to the gene product of the negative selection marker gene and
(b) separating the cells containing the bound binding molecule.
Substances are used as binding molecules which can bind specifically and preferably with high affinity to the negative selection marker. Preferab
Auer Johannes
Honold Konrad
Sprenger Raimund
Arent Fox Kintner & Plotkin & Kahn, PLLC.
Gansheroff Lisa
Ketter James
Roche Diagnostics GmbH
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