FI108729B - Method for expression of non-selectable genes in eukaryotic cells, which are not DHFR cells - Google Patents

Description

108729

The present invention relates to a method for expressing non-selectable genes in eukaryotic cells which are not DHFR 'cells. DNA transfection is a widely used method for transferring foreign genes into animal cells in culture. In recent years, this technique has enabled, on the one hand, the identification and analysis of a substantial number of regulatory regions of 10 different genes and, on the other hand, the production of pharmaceutically interesting proteins in their natural or substantially natural form.

Currently, quite a number of different techniques can be used for gene transfer. According to the technique used, the transferred genes integrate as single copies or as associated concatamers in the statistical process into the host cell chromosome. The degree of expression of the transducer (s) y (s) gene (s) will then depend on a number of factors: e.g., copy number, integration site, promoter used, ·. tor or enhancer potency and mRNA stability.

At the present state of the art, the construction of foreign DNA within the '··' circuit of a chromosome with high transcriptional activity 'is still a largely unresolved problem. Copy number However, can be manipulated. Because of the relationship between copy number and expression level, systems that ·; · *: allow for gene amplification are of interest so that 30 genes can be expressed at high yield. Over the past few years, you have described a set of amplifiable eukaryotic genes. Among the latter, the dihydrofolate re- ductase gene (DHFR), which is amplified in cells in response to the increased concentration of methotrexate inhibitors, is best described. "Chinese Hamster Ovary" (CHO) cell line, which no longer has an endogenous I 2 108729 j DHFR gene (Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci.

USA 77, 4216-1220), is thus particularly suitable for amplification of the exogenous DHFR gene and its associated gene (Kaufmann and Sharp, 1982, J. Mol. Biol. 159, 601-621).

However, this method is very laborious and time-consuming to "screen" a good productive cell line, as sufficient amplification and expression usually requires 4-8 months. In addition, the amplification from MTX to the CHO system or a similar cell line 10 has so far been limited, as it is often followed by non-specific resistance in other non-DHFR deficient cells. In addition, competitive amplification of the innate DHFR gene cannot be ruled out, potentially shunting the desired amplification power.

The method of the invention for expressing non-selectable genes in eukaryotic cells other than DHFR "cells is characterized in that two or more selection marker genes, which are a neomycin resistance-mediating gene, a puromycin-resistance Gene-20 gene, or , transfected with non-selectable genes, wherein the selectable marker genes and non-selectable genes are one to one; and vectors or DNA constructs, and after that all transfected selectors are selected.

The present invention is a direct and rapid "screening" method of a highly productive cell line or wild type cell line of any origin:. With this method, two _ · t selectable genes are first transferred to the corresponding cells or wild type small cells with the 30 non-selectable gene (s) of interest. It is known that co-transfection * · is a very effective method for the simultaneous delivery of separate genes into animal cells. Co-transfection-35 also includes, very commonly, transfection of vectors and DNA constructs. Simultaneously transferred DNA-3 108729 fragments usually integrate directly into one another on the host chromosome and in many cases are expressed by high correlation. Subsequently, resistance to all markers is selected by simultaneously or briefly adding, in the first step, the corresponding (blocking) agent to the medium. Simultaneous addition or addition within one hour is more preferred, but the invention also extends to increments between 1 and 120 hours. This results in cell line 10s having a markedly increased expression level for a non-selectable gene compared to cells selected for only one marker gene. Furthermore, not only an additive but a multiplicative effect on the expression obtained is obtained. The use of two or more selectable markers in this system avoids difficulties that depend on non-specific resistances. In addition, said "marker genes" may be shared with the same vector or shared with multiple vectors with the non-selectable gene of interest.

Further, it is now possible, by increasing the concentration of one (more) selectant in the culture medium, while keeping the concentration of the other (s) constant, to select all the transfected genes, and especially the high expression level V: non-selectable gene.

The advantage of this method is its wide applicability to any cell line and the avoidance of long-term screening procedures; Thus, e.g.

30 protein mutants are genetically engineered in the short term to produce the amount required for efficacy testing and subsequently tested.

The invention thus relates to the above-mentioned method for the expression of highly expressed recombinant ____; 35 to produce mutants as well as its use to search for mutants carrying a non-selectable gene. Further developments of the invention are set forth in the following examples and claims.

Examples 1. Double lection of "Baby Hamster Kidney" (BHK) cells

The number of transfected genes in the simultaneous transfection method described above is not a limiting factor, so that more than two genes can be introduced into a cell simultaneously. As an example of the simultaneous transfection of human an-10 thrombin III transcription unit with various selectable marker combinations (neomycin resistance / DHFR; neomycin resistance / puromycin resistance) into BHK cells, the dual selection system of van heterologous structural gene, advantageous properties.

1. a) Co-transfection of pSVATIII with pAG60 / pADHFR (neomycin resistance / DHFRcDNA) 20 BHK 21 (ATCC CCL-10) cells increased this co. for Dulbecco Modified Eagles (DME) medium containing 10% newborn calf serum (NCS).

"!! For transfection, the following circular plasmid DNA was co-precipitated with calcium phosphate texyl 25 (Graham and van der Eb (1973) Virology 52, 456-V '467) to 0.5 ml: 2 pg pSVATIII (Zettelmeissl et al. (1987) ', Biotechnology 5, 720-725), 0.8 pg pAdDHFR (Kaufman and

Sharp, 1982, a.a.O.) and 0.2 pg of pAG60 (Colbe're-Garapin et al. (1981) J. Mol. Biol. 150, 1-14). The precipitate was placed with 30 5 ml medium on 1.2 x 105 cells in a 25 cm 2 culture vessel and incubated for 16 h at 37 ° C. After an additional 48 h incubation of cells in 5 ml DME / 10% NCS, cells were transferred to 1: 3 DME / 10% NCS medium + 500 pg / ml G418 (Geneticin, Gibco) + 0-300 nM MTX After 6-14 days of selection time (medium change every 3 or 4 days), resistant colonies were counted, trypsinized, and added to t 5 108729 as a clone mixture (mixed clone, meaning collection of the resulting clones).

To determine the degree of AT ΙΙΙ expression, 5 x 10 -5 cells from such a mixed clone were placed in 25 cm 2 culture dishes and 5 ml medium. After 24 hours, fresh medium was added for another 24 hours.

Cells were counted and the AT III concentration determined by a specific Enzyme Linked Immunosorbent Assay (ELISA) (proposed in DPA P3624453.8).

Figure 1 shows that the number of clones per transfection decreases with increasing MTX concentration, against which the amount of secreted AT II1 in double selection is four times higher than by simple selection with G418.

Chromosomal DNA was isolated from different MTX concentrations, double-select BHK clones and hybridized by Dot-Blot (Maniatis et al. (1982) Molecular Cloning - A Laboratory Manual. Cold Spring Harbor, 158, 393-401). translation with a 32 P-labeled AT III cDNA fragment. The results show that increasing resistance to MTX correlated the average AT III gene copy number of the clone mixture! 'h'. 'and the degree of AT ΙΙΙ expression.

B) Simultaneous transfection of pAB3-1 plasmid (AT III) with plasmids pRMH140 / pSV2dhf r (neomycin V: resistance / DHFRcDNA):, v This example should show that the system shown in Example 1a) with a slightly modified protocol ·; · * Can also be performed on vectors having other transcriptional control sequences for each gene (AT III, t · (neomycin resistance, DHFR).

* # · “· '· For this experiment, BHK cells were grown in DME-' 'medium containing 10% fetal calf serum (FKS) (medium). For transfection, the following circular plasmid DNAs were co-precipitated with calcium phosphate (ω) to a volume of 1 ml at ____: 35: 20 pg pAB3-6 108729 1.5 pg pSV2dhfr (Lee et al. (1981) Nature 294, 228-232) and 5 pg pRMH140 (Hudziak et al. (1982) Cell 31, 137-146).

pAB3-1 consists of the plasmid pSVATIII, which has an "Enhancer region" ligated to its EcoR1 site at the human cytome! 5 galoviruses, paragraphs 147-598 (Boshart et al. (1985) Cell 41, 521-530). The precipitate was directly applied for 30 minutes at 37 ° C to 5 x 105 cells in a 25 cm 2 culture vessel. Growth! 5 ml of media was added and the cells were allowed to incubate for an additional 5-6 hours at 37 ° C. Cells were then treated for 3 minutes at 37 ° C with 15% (v / v) glycerin in culture medium. After washing the cells twice with culture medium, they were incubated for 72 hours at 37 ° C in culture medium. For selection, cells were subsequently transferred in a 1: 3-1: 4 medium containing 400 pg / ml G418 with or without 1 μΜ MTX.

After a selection period of 10-15 days (changing media every 3-4 days), resistant colonies were counted, trypsinized and allowed to multiply as a clone mixture. The level of AT III expression was determined as described in Example 20a1a).

Table 1 shows that the number of clones generated by double selection per transfection is about five times lower than when selecting with G418 for one-; other. Furthermore, it is evident that the AT III-25 expression levels after double selection are about V 'six times higher.

Table 1 ! G418 MTX Colonies / # ATIII Expression # ·; [pf / ml] [μΜ] Transfection [pg / 106 cells / 24 h] 30 _, ·, 400 0 300 ± 30 1 ± 0.2 ′ · 400 1 60 ± 15 6 ± 0.3 ____: 35 # Triple mean transfection assay mean 7 108729

The double-selected mixed clone (3MK1; see Table 2) was gradually exposed to higher MTX concentrations in the medium (1-10 μΜ). Doubling of the AT III expression level could then be achieved. The mean copy number of AT III as determined by the titanic Southern blot method of Kwan-5 (Zettlmeissl et al., A.a.O.) likewise increases with this secondary MTX selection (Table 2).

10 Table 2 G418 MTX ATIII Gene Copy- ATIII Expression [μί / ml] [μΜ] ta / Cell (3MK1) [μg / 106 cells / 24 h] 15,400,40 6 ± 0.2,400 10,150 12 ± 0, C) Co-transfection of pSVATII1 with 20 plasmids pAG60 / pSV2PAC (neomycin resistance / -. Puromycin resistance) and BHK cells were co-transfected as

Example 1a) is described in which 0.8 pg of pSV2PAC was used in place of pAdDHFR (Vara et al., 1986, Nucl. Acids Res. 11, 4617-4624). (pSV2PAC encodes · N-acetyltransferase of Streptomyces alboniger and gives resistance to puromycin antibiotic when expressed in animal cells.) Transfected cells were selected ·· ** · DME / 10% in NCS medium + 500 pg / ml G418 + 0-30 pg / ml of puromycin, allowed to multiply as a mixed clone, and as described above, tested for its AT III expression.

'· * In this experiment too, the number of clones obtained per transfection increases with increasing puromycin concentration. In contrast, AT III expression levels are six-fold higher in double-35-selected cells than in cells that have been selected for G418 alone (Fig. 2).

8 108729 2. Double selection in other cell systems

The following examples are intended to demonstrate that the dual selection principle can also be applied to animal cells in culture.

2. a) Co-transfection of pSVtss + plasmid (containing cDNAs for human interferon-β) with pAG60 / pSV2PAC (neomycin resistance / puro-mycine resistance) in CHO and mouse LKK cells.

CHO dhfr ~ (Urlaub and Chasin (1980) aaO) and L10 TK '- (ATCC CCL1.3) cells were co-transfected as in Examples 1a) and le) described by the following circular plasmid DNA: 2 pg pSVtss ( Reiser and Hauser (1987) Drug Res., 37, 482-485), 0.2 pg pAG60 and 0.8 pg pSVPAC. After selection, DME / 10% NCS + G418 (360 pg / ml for 15 CHO cells; 700 pg / ml for 1 TK 'cells) + puromycin (0-10 pg / ml) was counted for the number of stable transfectants, cells allowed to proliferate as a clone. and the degree of interfero-ηί-β expression was determined in an antiviral assay (Finter (1969) J. Gen. Virol. 5, 419-427). In addition, confluent cells were treated with ροζ, ly (I): poly (C) for 5 hours. For the test, 15 hours of oral spleen cells were collected (Dinter and Hauser (1987) EMBO J. 6, 599-604). The CHO co-clones, double-selected with 10 'pg / ml puromycin and G418, expressed 20-fold more interferon-β than those subjected to only one G418 selection (Fig. 3A). 1 TtC mixed clones double-selected with 10 pg / ml puromycin and G418 expressed approximately 6-fold more interferon-β than those with only one. G418 selection (Fig.

30 3 B).

/, 2. b) Co-transfection of pAB3788 (containing cDNA in human '* Factor VIII: C) with pRMH140 / *' * PSV2dhfrplasmids in HE7 cells (hamster) 108729

To construct plasmid 3788, a cDNA was cloned; which encodes a deletion mutation of human Factor VIII: C (deletion at amino acids 741-1689, as proposed in German Patent Application P 37 20 246.4) and has a Xho I site on both sides of the vector pAB3-1 (see Example Ib). In addition, pAB3-1 was cut with SalI / XbaI, the overhanging 5 'ends were filled with the E. coli Polymerase I Klenow fragment and purified as a vector fragment (3.3 kb). After the overhanging 5 'ends of the XhoI site of the purified Factor 10 VIII: C DNA fragment were similarly filled in, vector pAB3788 was obtained by ligation. 20 µg of pAB3788 as described in Example Ib) were co-transfected with pRMH140 and pSV2dhfr in HE7 cells (Cook, J.L. and Lewin, A.M. 15 (1979) Cancer Res. 39, 1455-1461). The selection described in Example Ib yielded about 300 ± 30 clones per transfection. The transfectants as a co-clone expressed 0.7 + 0.1 units (U) of Factor VIII: C per 106 cells for 24 hours. The double-selected cells, Ekspres-20, thus exhibit a factor of 3 to 6 more than the Factor VIII: C, · mutants than the mixed clone selected in the G418 independent assay. Factor VIII: Activity was determined by the COA test (Kabi Vitrum, Uppsala, Sweden).

3. Double selection as a method for obtaining a high recombinant * retinovirus particle titer V: Retrovirus expression vector pM5AT III construct

The 1.4 kb BglII / BamHI fragment (AT III

cDNA) to the soluble BamHI site of pM5neo flanked by a ··· myeloproliferative Sarcoma virus cleavage donor site or cleavage acceptor site. The ATIII fragment / (consisted of the attachment of the BglII linker to the 1.6 kb SalI / Eco RI fragment of the vector 's; pSVAT, followed by BamHI / BglII digestion. PM5neo has a pBR backbone in addition, among others, junctions and the neomycin-35 resistance gene as well as myeloproliferative sarcoma mavirus LTRs (Ostertag et al.

10, 08729 (1980) J. Virol. 33, 573-582; Stacey et al. (1984) J. Virol. 50 725-732).

The helper cell line Psi2 (Mann et al. (1983) Cell 33, 153159) was allowed to proliferate in DME with 10% FKS5. For transfection, a mixture of circular plasmid DNA (5 pg pM5AT III and 2 pg pSV2PAC) and 8 pg digested DNA from mouse L cells was precipitated with calcium phosphate in a volume of 0.5 ml and incubated for 30 minutes at room temperature. x 105 on Psi2 cell in 10 5 ml media. After approximately 12 hours of incubation at 37 ° C, the medium was changed and again 24 h later replaced with selection medium (single selection 1 mg / ml G418, double selection 1 mg / ml G418 and 2 or 5 pg / ml puromycin DME). platform plus 10% FKS). The resulting clone-15 nites were counted after 10-24 days and collected.

For the virus titer assay, 5 x 105 were seeded

Psi2 cells and medium were changed after 24 hours. 24 hours later, supernatant containing recombinant retroviral particles, or equivalent dilutions, was used for infection of HIH 3T3 cells (ATCC CRL 1658; 1000-3000 cells, · 24-well plate). The infection occurred under 8 µg / ml polybrene addition for about 12 hours.

* ;;; Subsequently, the medium was changed and, after 24 hours, was replaced with selection medium (1 mg / ml G418). The resulting clones * ··· * 25 were stained and counted 10 days after selection medium exchange V '. Clonal mixtures resulting from double selection ·, ·, · * achieve approximately 10 times higher titer of living cells than single selection selected: ··· (Fig. 4).

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»4 · t» »I · •» · I | I I · · • · »n 108729 i Explanations Figure 1-3

The number of stable transformants is plotted in left order and shown in open panel sizes. The gene product of interest (AT

! III or interferon) is represented by the right order herein and is represented by open circles. The abscissa shows the concentration of the respective variable inhibitor (methotrexat or puromycin).

I · »li · • ·» I i. . * '· *

Claims (6)

A method for expressing non-selectable genes in eukaryotic cells that are not DHFR-cells, characterized in that two or more selection marker genes which are a neomycin resistance-mediating gene, a puromycin resistance-mediating gene or a DHFR gene, is transfected with the non-selectable genes, wherein the selection tag genes and non-selectable genes are located in one or more vectors or in the DNA structure, and then selected on all transfected selection tags. Method according to claim 1, characterized in that CHO, BHK, LTK or HE7 cells are used. 15 Method according to claim 1, characterized in that a single, non-selectable gene is co-transfected. Method according to claim 1, characterized in that in the following selection steps the concentration of one or more inhibitors, preferably an inhibitor, is increased. 20 with the factor 2-100, preferably with the factor 8-12. 1 The method of claim 1, characterized in that the non-selectable gene encodes AT III, F VIII: C, F XHIa, t-PA, EPO, G-CSF, GM-CSF, PAI, protein CII. '!! or IL-3. 1 * *. 25 The method of claim 1 for searching for * * * mutants of non-selectable genes. * i »» #> »» r l i t I * i t t I I I! ; in