Positive-negative selection for homologous recombination

The invention concerns a method for the introduction of a foreign DNA into the genome of a target cell by homologous recombination as well as for the homologous recombination of suitable DNA constructs.

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.sup.- 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. Preferably
 those binding molecules are used which do not have any interfering
 cross-reactivity with other surface components of the an host cell.
 Examples of binding molecules are antibodies e.g. polyclonal or monoclonal
 antibodies, antibody fragments etc. which are directed against the gene
 product of the negative selection marker gene. Suitable antibodies to
 dLNGFR are for example known from WO 95/06723. When a receptor is used as
 a negative selection marker, a natural binding partner of the receptor,
 e.g. the receptor ligand or an analogue thereof, can of course also be
 used as a binding molecule. An example of such a receptor ligand is NGF as
 a ligand of LNGFR.
 In order to facilitate the separation of the cells labelled with the
 negative selection marker, it is possible to use a binding molecule which
 is coupled to a solid phase and this coupling can be achieved by
 adsorption, covalent binding or by a high-affinity binding pair (e.g.
 streptavidin/biotin). The type of solid phase is generally uncritical for
 the method according to the invention and preferably those solid phases
 are used which enable an easy separation of the cells presenting the
 negative selection marker from unlabelled cells. Therefore the solid phase
 can be for example present in the form of a chromatographic column, but
 particulate solid phases such as microbeads, in particular magnetic
 microbeads, which enable a particularly simple separation are especially
 preferred.
 Alternatively the transfected cells can also be contacted with free binding
 molecules. In this case the free binding molecules preferably carry a
 marker or/and a solid phase binding group. Examples for suitable marker
 or/and solid phase binding groups are biotin, biotin derivatives, e.g.
 iminobiotin, aminobiotin or desthiobiotin, haptens, e.g. digoxigenin,
 fluorescein, enzymes e.g. peroxidase or alkaline phosphatase or dyes e.g.
 fluorescent dyes such as fluorescein, phycoerythrin, rhodamine,
 peridinine-chlorophyl protein, Texas red or derivatives thereof.
 If a binding molecule is used which carries a solid phase binding group
 such as biotin, a biotin derivative or a hapten, the cells labelled with
 the binding molecule can be coupled to a solid phase that can react with
 the solid phase binding group of the binding molecule. If a binding
 molecule is used which carries a biotin group, one can for example
 identify the cells expressing the negative selection marker and separate
 them from unlabelled cells by binding to an avidin or streptavidin-coated
 solid phase.
 If a binding molecule is used which carries an enzymatic marker group, the
 cells expressing the negative selection marker can be identified after
 addition of an enzyme substrate by an enzyme catalysed color reaction and
 optionally separated from unlabelled cells. This identification can for
 example be carried out by putting the cells on a slide and subsequently
 analysing them microscopically.
 If a binding molecule is used which carries a fluorescent dye, the cells
 expressing the negative selection marker can be identified by flow
 cytometric analysis and separated from unlabelled cells. This separation
 procedure is rapid and simple and can be carried out in conventional FACS
 instruments that enable the setting of fluorescence windows and cell
 sorting.
 A further subject matter of the present invention is a recombinant vector
 which is suitable for use as a transfection vector in the method according
 to the invention. This vector comprises:
 (a) two flanking nucleotide sequences that are homologous to a target
 sequence in a cell,
 (b) a nucleotide sequence coding for a positive selection marker under the
 control of an expression control sequence that is active in the cell and
 which is located inside of the two flanking sequences according to (a),
 (c) a nucleotide sequence coding for a negative selection marker under the
 control of an expression control sequence that is active in the cell which
 is located outside the flanking homologous nucleotide sequences and whose
 expression product is a polypeptide located on the cell surface.
 If it is intended to use the recombinant vector to activate an endogenous
 gene in the host cell, it contains an additional heterologous expression
 control sequence which is active in the host cell between the two flanking
 homologous nucleotide sequences. This expression control sequence
 comprises a promoter and preferably further expression-improving sequences
 e.g. an enhancer. The promoter can be a regulatable or a constitutive
 promoter. The promoter is preferably a strong viral promoter e.g. an SV40
 or a CMV promoter. The CMV promoter/enhancer is particularly preferred.
 If an amplification of the target gene in the transfected host cell is
 desired, the recombinant vector contains an amplification gene between the
 two flanking sequences. Examples of suitable amplification genes are
 dihydrofolate reductase, adenosine deaminase, ornithine decarboxylase etc.
 A particularly preferred amplification gene is the dihydrofolate reductase
 gene, in particular a gene coding for a dihydrofolate reductase arginine
 mutant which has a lower sensitivity towards the selective agent
 (methotrexate) than the wild type polypeptide (Simonsen et al., Proc.
 Natl. Acad. Sci. USA 80 (1983), 2495).
 The nucleotide sequence coding for the negative selection marker can--as
 elucidated above--preferably be selected from membrane-based receptors or
 membrane-based receptor fragments containing the ligand binding domain of
 the respective receptor.
 The flanking nucleotide sequences that are homologous to a target sequence
 can be selected from any chromosomal regions of the genome of the cell to
 be transfected which is preferably a eukaryotic cell, particularly
 preferably a mammalian cell and most preferably a human cell. In the case
 of human cells the flanking homologous nucleotide sequences are preferably
 derived from the region of genes for human factors e.g. EPO, tPA, G-CSF,
 GM-CSF, TPO, interleukins, interferons, growth factors, insulin,
 insulin-like growth factor etc.
 The flanking homologous nucleotide sequences can include the coding region
 of the target gene or a part thereof. In this part they can be selected
 such that in a homologous recombination they cause a mutation in the
 coding region of the mature target polypeptide compared to the endogenous
 sequence present in the cell. This mutation can comprise substitutions,
 deletions and insertions of individual amino acids or whole amino acid
 sections.
 Yet a further subject matter of the present invention is the use of
 membrane-based surface receptors as negative selection markers in a method
 of homologous recombination.

EXAMPLES
 Methods
 Recombinant DNA Technique
 Standard methods were used for the manipulation of DNA as described in
 Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual,
 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The
 molecular biological reagents were used according to the manufacturer s
 instructions.
 Transfection of Human Cell Lines, Cultivation and Cloning
 The vector was present dissolved at a concentration of 1 .mu.g/.mu.l double
 distilled water. In order to ensure a high transfection efficiency, the
 cells were transfected with the aid of electroporation (BioRad,
 Genepulser.TM.) under conditions that were-previously determined to be
 optimal (960 .mu.F/260 MV/18-22 .mu.S). The adherently growing human
 fibrosarcoma line HT1080 (ATCC CCL 121) was used as a suitable cell line
 at a concentration of 10.sup.7 cells/0.8 ml. The cells were kept for ca.
 10 min on ice before and after transfection in order to reconstitute the
 cell membrane.
 Transfected cells were sown in T-175 culture flasks and cultured in an
 incubator at 37.degree. C. and 7% CO.sub.2. After 24 h selection pressure
 was applied by adding G418 (0.8 .mu.g/ml).
 After 14 days in culture resistant clones appeared in the culture dish.
 After larger foci had grown, the cells were washed with PBS, trypsinized
 and stained as single cell suspension.
 FACS Analysis
 The staining steps were carried out on ice using 10.sup.5
 cells/preparation. The anti-dLNGFR antibody from the mouse used as the
 primary antibody was detected by adding a secondary antibody from the goat
 (a-mlgG-FITC, 1:25, Caltag). The cells were stained with the secondary
 antibody alone as a control for unspecific binding. Dead cells were
 detected by adding propidium iodide (10 .mu.g/ml). The analyses were
 carried out on a FACS-Vantage (Becton Dickinson Co.) according to the
 manufacturer's instructions. The specific fluorescence of cells expressing
 dLNGFR was recorded in the FL-1 channel and the dead cells in the FL-3
 channel.
 Example 1
 Preparation of the Expression Construct for dLNGFR
 The gene for dLNGFR (WO 95/06723, Boehringer Mannheim GmbH) which comprises
 965 bp was amplified with the aid of the PCR method. The primers used
 introduced cleavage sites for the enzymes EcoRI and SalI at both ends.
 After amplification the PCR fragments were cleaved with both enzymes.
 The vector pSV1 which contains the early SV40 promotor and the SV40
 polyA-signal (Okayama and Berg, Mol.Cell.Biol.3 (1983), 280-289; Muligan
 and Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072-2076) was also
 cleaved with EcoRI and SalI.
 The isolated vector has a size of 3490 bp. The dLNGFR fragment is ligated
 into the vector pSV1. The gene for dLNGFR was under the expression control
 of the early SV40 promoter and the SV40 poly-signal. The entire expression
 cassette comprises 1900 bp. The resulting vector PSV-DLNGR is shown in
 FIG. 2.
 Example 2
 Testing the Expression Cassette for Functionality
 Cells of the line HT1080 were transiently transfected with the plasmid
 pSV-DLNGFR as described above. After two days growth the cells were
 analysed for expression of dLNGFR with the aid of the monoclonal
 anti-dLNGFR antibody. The result is shown in FIG. 3 which shows that
 dLNGFR-expressing and non-expressing cells can be distinguished by FACS
 analysis. It also shows that the reaction of the anti-dLNGFR antibody is
 specific for transfected cells.
 Example 3
 Cloning the dLNGFR Expression Cassette Into a Gene Targeting Vector
 The dLNGFR expression cassette was isolated from pSV-DLNGFR using the
 restriction enzymes Notl and Pvull. The targeting vector `p187` for the
 human EPO-gene (described in EP 97 112 649.5 and EP 97 112 640.5 see FIG.
 4b) was cleaved with NotI and EcoRV. The 14551 bp large vector fragment
 was isolated and ligated with the dLNGFR expression cassette (FIG. 4). The
 resulting plasmid `p187-DLNGFR` was transferred into E.coli and propagated
 therein.
 Example 4
 Test for Negative Selection in the FACS Scan
 HT1080 cells were transfected with p187-DLNGFR and selected for stable
 integration i.e. G418 was added to the medium 24 hours after transfection.
 The first FACS analysis was carried out after ca. 3 weeks growth and
 namely after formation of the first foci whose cells were pooled. As shown
 in FIG. 5 dLNGFR negative cells, in this case 14% of the population, can
 at this time be distinguished from the dLNGFR-expressing cells by FACS
 analysis.
 In addition to the rarely occurring event of homologous recombination this
 cell population, also contains cells whose surface receptor density is too
 low and therefore are not recognized by the detection system. However, in
 this manner the number of clones which have to be subsequently tested for
 the expression of the target gene can be considerably reduced (in this
 case 14 of 100%).
 If in a transfection preparation no clone is present which contains a
 homologously recombined targeting vector, then this situation can be
 indicated with much less work compared to the conventional screening. The
 absence of homologously recombined clones is demonstrated by the
 occurrence of a population reacting 100% with anti-dLNGFR antibodies. In
 this case it is not necessary to screen further for the expression of
 target gene.