Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

• Selective genetic markers for eukaryotic cells process for using such markers and application of cells containing such a marker to the production of proteins determined after their transformation with a corresponding DNA.
• the invention relates to a new, dominant genetic marker capable of penetrating a wide variety of eukaryotic cells, more particularly those originating from higher organisms and of giving them a stable specific character allowing the easy detection of the cells thus transformed, when these these are placed in specific culture media.
• the invention therefore also relates to a method for detecting eukaryotic cells to which a particular character has thus been conferred, as well as the application of such cells to the production of specific proteins, prokaryotes, eukaryotes or virals, when these cells have previously been transformed by a foreign DNA fragment containing a DNA sequence, such as a gene or cDNA coding for said protein.
• the enzymatic activities induced by these DNAs more particularly thymidine kinase and adenine phosphoribosyl transferase can be detected easily.
• the techniques used remain difficult to implement, however, since they involve the use of cultures of cells having characteristic mutations, in particular cells which have undergone an induced or induced mutation affecting the corresponding gene.
• the detection technique using a herpes virus thymidine kinase gene can be carried out in type L mouse cells first modified by an induced or induced mutation affecting their endogenous thymidine kinase genes , as already described by M. WIGLER et al. ("Cell.”, Vol. II, 223-232, 1977).
• TK cells thymidine kinase genes
• HAT medium containing hypo ⁇ anthine, aminopterin and beyond. thymidine
• mutant cells used in the labeling tests generally do not have the stability character of mouse TK- L cells. The character acquired by the mutation produced is quickly lost during the successive cell divisions of the cells in question.
• transposen of signs DNA elements of which many representatives are well known, which contain a gene coding for a "transposase”, allowing them as well to integrate into a first DNA, in particular that of the genetic heritage of a first type of cells , than detaching from it to transpose or incorporate into another DNA, for example that of a bacteriophage.
• the object of the invention is to make eukaryotic cells, and more particularly to those originating from higher organisms, applicable to labeling techniques involving resistance genes or analogs to those of antibiotics which, such as G418, are revealed or will prove to be capable of inhibiting the development of these eukaryotic cells.
• the invention naturally also has for its object the methods using such markers, as well as the application of the cells thus labeled to the use of cultures producing particular proteins, after their prior transformation by a DNA containing the sequence coding for such a specific protein.
• the marker according to the invention consists of DNA, circular or not, containing a eukaryotic promoter or recognized by its capacity to direct the expression in a eukaryotic cell of a gene which is normally associated with it and a DNA sequence coding for an enzyme capable of inactivating an antibiotic, such as the antibiotic G418, linked to this promoter and under its direct control.
• the DNA sequence coding for the inactivation enzyme is chosen from those which code for an aminoglycoside 3'-phosphotransferase
• the process according to the invention for labeling eukaryotic cells is characterized by bringing these cells into contact with the marker according to the invention under conditions suitable for allowing the transformation of at least part of these cells and for putting them in culture in a medium suitable for their development and containing an antibiotic, such as the antibiotic G418, normally toxic towards these cells and capable of being inactivated by the inactivation enzyme, in particular an aminoglycoside 3'-phosphotransferase synthesized by the above DNA sequence '.
• an antibiotic such as the antibiotic G418, normally toxic towards these cells and capable of being inactivated by the inactivation enzyme, in particular an aminoglycoside 3'-phosphotransferase synthesized by the above DNA sequence '.
• the marker according to the invention comprises, on the one hand, a part originating from a prokaryotic DNA, such as phage or preferably plasmid comprising an origin of replication allowing its cloning in a bacterial culture, and, on the other hand , incorporated into this part of prokaryotic DNA, the aforementioned eukaryotic promoter or capable of directing the expression of an associated gene in a eukaryotic cell, as well as said DNA sequence coding for the inactivation enzyme, in particular an APH ( 3 ') under the direct control of this promoter.
• a prokaryotic DNA such as phage or preferably plasmid comprising an origin of replication allowing its cloning in a bacterial culture
• the promoter is that under the control of which the transcription of the thymidine kinase gene is exerted in the cells from which it originates, and the gene coding for APH (3 ′) -II is a gene for resistance to kanamycin and / or to neomycin, such as that carried by the transposon well known under the designation "Tn5".
• promoters such as those of the genetic markers previously used and which have been identified above, or alternatively promoters belonging to viruses known for their ability to infect the eukaryotic cells used, for example.
• promoters such as those of the genetic markers previously used and which have been identified above
• promoters belonging to viruses known for their ability to infect the eukaryotic cells used for example.
• any sequence capable of synthesizing an enzyme endowed with inactivation properties vis-à-vis the antibiotic which will be used may be used.
• the culture medium used to operate the selection of the cells in which the genetic marker will have been incorporated in the case where the antibiotic incorporated in the medium is constituted by the antibiotic G419, it is advantageous to have recourse to phosph ⁇ rylatlon enzymes of the APH type (3) already mentioned. It goes without saying that one can have recourse to all other types of DNA sequences synthesizing an enzyme having a similar inactivation capacity of the antibiotic, for example by acetyiation of the corresponding hydroxyl functions of the antibiotic.
• the DNA sequence of the genus in question can come from any source appropriate.
• the advantage of having more specifically the sequence coding for the enzyme will result from the explanations given below, as regards the preferred genetic markers of the invention.
• the first nucleotide pairs of the DNA sequence coding for APH (3 ') or the like are made as close as possible to the last pairs of promoter nucleotides in the direction of transcription, in particular at a distance, expressed in number of pairs of nucleotides, less than 1,000, more preferably 500, or even less than 100 pairs of nucleotides. It has in fact been observed that the reduction of this distance, in particular by the use of appropriate deletions, has "the effect of a considerable increase in the transformation yield, as will appear during the description below of preferred markers conforming to the invention.
• the marker DNAs of the invention also contain, downstream of the APH gene (3 ′), a polyadenylation site, this latter being preferably also made as close as possible, to the last pair. of nucleotides of the gene coding for APH (3 ′).
• the distance expressed in number of base pairs between the end of this latter gene and either the polyadenylation site or the "stop codons" for translation of this gene is less than 1,000, this preference at 500 base pairs or even less than
• the labeling yield can be further increased if the marker DNA is given a eukaryotic origin of replication by incorporation of a fragment containing it, such as, for example, repetitive fragments of the DNA of the virus known under the designation " Saimiri herpes virus ".
• a fragment containing it such as, for example, repetitive fragments of the DNA of the virus known under the designation " Saimiri herpes virus ".
• Saimiri herpes virus The existence of episcmal forms of this virus and DNA subunits of 1.4 kilobases in multimeric forms in corresponding defective genomes suggests that these subunits contain an origin of replication.
• Preferred markers according to the invention are obtained by modification of the plasmids pAGO or pFG5, themselves derived from pBR322.
• the plasmid pAGO is preferred in that it comprises, in addition to a TK gene, two additional resistance factors to ampicillin and to tetracycline.
• the structure of pAGO is shown schematically in Figure 1 of the drawings.
• the TK gene is notably represented therein schematically by a circular arc in broken lines and various restriction sites more particularly taken advantage of in the construction of preferred markers in accordance with the invention, the description of which will be given below.
• the arrow T of the TK gene gives the direction of transcription.
• the point O corresponds to the EcoRI site of this plasmid.
• the EPR and PAS sites correspond to the positions of the region of the promoter of the TK gene and of the site of polyadenylation of the TK gene respectively:
• the preferred genetic markers according to the invention are those which result from the recombination of pAGO and of the transposon Tn5 (described by JORGENSEN, HA, ROTHSTEIN, SJ and REZNIKOFF, V;. S. (1979), "Kolec. Gen. Genêt.” 177, 65-72), whose figure 2 provides a schematic representation and which were obtained from the bacterial plasmid ColEl Tn5 contained in the strain of E.
• the preferred antibiotic used in the above-mentioned detection methods is certainly constituted by the antibiotic G-418, the ability of which has been recognized to penetrate into most eukaryotic cells and destroy them by a toxic process. It goes without saying that another antibiotic can be used, provided that it proves to be toxic in the same way with regard to the type of eukaryotic cells which will be used in the detection process. will then be applied under the conditions of the invention.
• the corresponding marker must consequently contain a DNA sequence capable of synthesizing an inactivation enzyme capable of more particularly inhibiting the antibiotic chosen. Additional characteristics of the invention will become apparent during the description of preferred markers whose constitutions and constructions schematically result from Figures 2 to 13 of the drawings.
• the bacterial strains used for the transformations were E. co li 1 1 06 (803r- k m- k ) and 1107 (803r- k m + k ) (MURRAY, NE, BRAMHAR, WJ and MURRAY, K. ( 1976), "Molec. Gen. Genêt.”, 1 50, 53-61).
• the eukaryotic strains used were 1D clones of L TK- mice, Vero chimpanzee cells, MKO cells and HeLa human cells. The cultures of these cells were carried out in an EAGLE medium modified by DULBECCO, supplemented with 5 or 10% calf serum.
• the transformed cells expressing the enzyme APH (3 ') - II were selected in a medium containing from 10 to 400 micrograms per milliliter of the antibiotic G418.
• the TK cells were selected from the HAT medium, also under the conditions described by COLBEREGARAPIN and Coll. For the cloning of the cells, the individual colonies were isolated at random and cultivated until producing confluent cultures.
• the promoter used comes from the "eukaryotic promoter region" originating from the HSV1 TK EPR gene (hereafter called EPR) and which in the plasmid pAGO is located between the PvuII and Hindi site at the start of the TK gene (FIG. 1).
• EPR comprises at the 5 ′ end of the gene two TATA regions (TATA boxes), one of which is now known to play an important role for the transcription of the TK gene (MC KNIGHT, SL (1930) Nucl. Acids Res., 8, 5949-5964).
• a recombinant is produced by ligation on the one hand of a DNA fragment HincII-HincII of 2480 base pairs in which is incorporated the gene for resistance to kanamycin (schematically represented by the arrow "kana r ”) which normally contains transposon Tn5, which has been shown diagrammatically in FIG. 2, and, on the other hand, the plasmid pAG0 opened at the Hindi site by partial digestion in the presence of this restriction enzyme.
• the recombinant obtained was used for the transfection of a strain of Es che r ich ia co li 1106 (803 r- k m k -) the colonies resistant to the three antibiotics are kanamycin, t.tracycline and 1 'ampicillin.
• Two recombinant plasmids, pAG-40 and pAG45 were thus obtained. Restriction analysis shows that the 2480 base pair fragment. from Tn5 was incorporated into the HincII site located in the sequence TKla position 2301 de-pAGO (Fig.1). The orientation of this fragment was determined by analysis of these plasmids with the mcyen of restrictions by Xhol and EcoRI.
• the Xhol site of Tn5 is located near the 5 'end of the APH (3') - II gene. It can be seen (FIG. 3) that the Xhol site is very close in pAG-40 to the Hindi site, itself contiguous to the TK EPR region. On the contrary, the fragment is in the opposite direction in the plasmid ⁇ AG45.
• the plasmid pAG40 was therefore recognized as suitable for expression in eukaryotic cells. This plasmid which turns out also contain the bacterial promoter of APH (3 ') - II was able to resist up to 60 ⁇ g / ml kanamycin.
• the distance in pAG40 between the EPR and the first tripletAUG of the gene coding for APH (3 ') - II is approximately 1500 base pairs.
• a new recombinant was formed between, on the one hand, a BglII fragment located in pAG40 between the coordinates 3670 and 4795 of its restriction map, and on the other hand pAGO, which had been open at the BglII site of the TK DMA fragment.
• the plasmids obtained, pAG50 (Fig.
• pAG55 which differ from pAGO by the presence of an insert of 1125 base pairs delimited by BglII ends, the closest to the latter being at a distance of about 130 pairs of bases of the EPR region.
• SmaI restriction by means of the enzyme SmaI and by analysis on gels, it was determined that the 5 ′ end of the APH gene (3 ′) - II was connected to the TK ⁇ PR, while the insert was integrated into the opposite direction in the plasmid pAG55. It is therefore the plasmid pAG50 which was chosen for obtaining the expression of the enzyme under the control of the regulatory region of TK.
• the molecular weight of pAG50 (like that of pAG55) is 7490 ⁇ 50 base pairs.
• the TK polyadenylation site (NOT in the drawings) is located in pAG50 Immediately downstream of the SmaI site in the direction of transcription of the TK gene (in the region of coordinates 4608 in Figure 4.
• the distance separating the 3 'end of the APH gene (3') - II and the TK polyadenylation site (McKNIGHT ) has been reduced by 1365 base pairs.
• the TK codc ⁇ tcp TGA is located at a distance corresponding to 21 base pairs downstream of the Smal site.
• the position of the stop codon of APH (3 ') - ÎI is not exactly known, but it is very close to it
• the new plasmid obtained has been designated by the expression pAG60 (FIG. 5) and has a length corresponding to approximately 6,150 base bones.
• pAG60 FOG. 5
• pAG60 pAG60
• the defective genome mentioned above of the DNA of the Herpes Saimiri virus was used, this genome of staff comprising a multimeric form of a subunit of 1.4 kilobases which is believed to contain an origin of replication.
• This workforce gene (from strain 11 of the Herpes Saimiri virus, described by Fleckenstein et al. (1979) Biochim.
• the antibiotic G-418 has an activity of synthesis of a large variety of inhibitory proteins. The toxicity of this antibiotic has therefore been tested against L mouse cells. TK-, clone ID, of chimpanzee cells (uncloned or cloneVCI0) and of OMK cells as well as with respect to human HeLa cells. The number of cells doubled in cultures in the first 48 hours after the addition of G-418. At a concentration of 25 ⁇ g / ml of antibiotic the ID cells did not become detached for a month. Using the usual antibiotic concentration (150 ⁇ g / ml) the ID cells died and detached from the plaques within 10 days.
• L TK- mouse cells The clone ID of L TK- mouse cells is highly sensitive to transfection, as shown by WIGLER et al. (1978) Cell, 14, 725-731). These cells were therefore chosen to study the transfer and expression capacity of the selective marker of the invention.
• the transfections were carried out as indicated above in bottles each containing approximately 3.10 6 cells.
• the selective agent G-418 was added 48 hours after transfection, at a concentration of 150 ⁇ g / ml. Colonies were counted three weeks after transfection. The results are presented in Table I below.
• the table shows that transformed colonies resistant to the antibiotic G-418 were obtained after transfection with the recombinant plasmids containing the APH (3 ') - II gene.
• Relatively low transformation yields of the order of 1 to 3 colonies per ⁇ g of DNA, were obtained with the bacterial transposon Tn5 and the plasmids pAG45, pAG40 and pAG55.
• pAG50 in which the TK promoter region has been brought much closer to the 5 'end of the APH (3') - II gene (20 to 30 colonies per ⁇ g).
• the antibiotic resistance phenotype G-418 is stable under the culture conditions used for the selection. It is the same again, even when the cells are cultivated in the same medium, but in the absence of the antibiotic. The cells do not become sensitive to the antibiotic G-418, even after several passes. Transformation by recombinant plasmids of monkey cells and human cells.
• the HSVl TK gene was used because of its easy detection capacity.
• the circularized plasmids pAG6 ⁇ and pFG24 carrying respectively the APH (3 ') - II and TK genes respectively were used in order to effect co-transformations or simultaneous transformations of TK-1D cells.
• the "two plasmids were used in mass proportions corresponding to 5 AFH (3 ') - II for 8 TK.
• This test is obviously representative of the possibilities offered by the selective genetic markers according to the invention, of locating eukaryotic cells transformed by another DNA, the expression of which could be sought, such as for example a DNA or cDNA coding for a protein. , such as insulin, human interferon, etc., or a viral protein intended for the production of vaccines, such as the HBs antigen of the viral hepatitis B virus, etc.
• the transformation yield and of detection is obviously likely to be more important if recourse is had, not to a co-transformation of the cells, as described above by way of example, with a marker plasmid conforming to invention and by a separate plasmid carrying the DNA sequence whose expression is sought, but to a transformation by the marker plasmid according to the invention itself, into which the tooth DNA sequence will have been inserted beforehandthe expression is searched.
• This last insertion can, in the case of the preferred plasmids which have been described above, be carried out with advantage in one of the sites containing the factors of resistance to tetracycline and to ampicillin, which they have in common with pAGO and which were not affected by the modification which led to the plasmids according to the invention.
• markers according to the invention For purposes of illustration, from the foregoing, a preferred application of the markers according to the invention will be described, more particularly their use as vectors containing an insert derived from a fragment of the genome of the viral hepatitis B virus, containing more particularly the S gene thereof and the genetic information to allow its expression in eukaryotic cells. Even if this is a preferred case, it is naturally understood that the invention cannot be limited thereto.
• FIG. 7 is in fact a representation of the plasmid pAG 60, therefore very similar to that indicated in FIG. 5.
• additional sites of restriction enzymes which will be used in the construction of the final marker.
• These are two Sph I sites located approximately 565 and 2,827 nucleotides respectively from the Eco RI site (represented by the letter “O” in the figures).
• the approximate position of an additional Pvu II site has been specified, in particular in the kanamycin resistance gene "kana r ", a site which is also used in the construction of the modified plasmid marker described below.
• FIG. 8 schematically represents a fragment delimited by Rsa I and Hinc II ends (hereinafter called Rsa I-Hinc II fragment), as it can be extracted from the plasmid pCP 10 described in European patent application No. 81,400,634, extraction which comprises the stages which consist in treating under conventional conditions said plasmid with the two corresponding restriction enzymes and in recovering the corresponding fragment, more particularly that comprising of the order of 820 base pairs and which happens to be provided with the above extremities.
• the Rsa I-Hinc II fragment of FIG. 8 is inserted into the Pvu II site located in the "kana r " gene of pAG 60, this genetic recombination being rendered particularly easy due to the blunt ends of the fragments to recombine, which is carried out under conventional conditions in the presence of a ligase.
• the resulting plasmid XAP I is shown diagrammatically in FIG. 9.
• the fragment represented in FIG. 10 is re-extracted and delimited, on the one hand, by the Sph I site which in the Rsa I-Hinc II fragment of the figure 8 was a short distance from the Hinc II end and on the other hand the Sph I site at the position of the order of 3 017 (opposite the Eco RI site identified by the sign "0" in the fig) of XAP I.
• the fragment obtained contains a large proportion of the S gene and of the genetic information necessary for its expression in eukaryotic cells.
• This fragment is then reinserted into the plasmid pAG 60, more particularly in its Sph I site at position 565, which leads to the plasmid pAG 61 of FIG. 11.
• this pla-smide does not contain all of the genetic information necessary for the expression of the S gene, it is preferable to carry out an additional genetic recombination of this plasmid with a complementary sequence extracted from DNA-HBV, represented in FIG. 12 and delimited by ends Xba I and Taq I at positions 2 938 and 1 276 according to the numbering of GALIBERT et al.
• Plasmid pAG 66 is thus obtained, which then contains a larger fragment of DNA HBV, extending from position 680 to position 1276 according to the numbering of GALIBERT et al (via site 2938).
• This marker plasmid containing an insert, itself containing the entire hepatitis B virus S gene as well as the genetic information necessary for its expression in eukaryotic cells is only one example of the numerous markers -vectors allowing the recognition of those of eukaryotic cells transformable by the latter. It goes without saying that all forms of inserts extracted from DNA HBV can be used, for example those described in European patent applications 81 400 634, 0 013 828; 0 020 251 and 81 00577. In particular, recourse may be had to the insertion of any sequence extracted from HBV DNA containing all the genetic information necessary for the expression of the S gene, however excluding the parts of HBV DNA which can be considered responsible for the infectivity of the viral genome.
• the invention applies naturally in the same way and under conditions analogous to the transformation and detection of higher eukaryotic cells transformed by a DNA or cDNA coding for any other determined proteins.
• the markers according to the invention are of very particular interest for the transformation of eukaryotic cells of higher organisms, more particularly animal or human cells, or also plant cells.
• the invention relates more particularly to the application of the markers as defined above and in which a nucleic acid fragment has been inserted comprising all the genetic information necessary for its replication and its expression in eukaryotic cells, to the production of the corresponding expression product, this application involving a process comprising the culture of the cells thus transformed and the recovery of said expression products, either from the cells, or from the medium when the expression product, in particular protein, is excreted in the culture medium.
• the invention lends itself in particular to the production of proteins having immunogenic properties, capable of inducing the production of antibodies, capable in turn of neutralizing the hepatitis B virus, and this all the more that a large part of the protein produced is excreted in the culture medium. It can be recovered from it by conventional purification techniques, using either physicochemical separation methods or immunological methods, for example affinity chromatographies on a substrate on which have previously been attached corresponding antibodies.
• the selective system which has been described has a number of particularly interesting characteristics and advantages.
• the selection does not imply the use of special metabolic pathways or nucleotide synthesis pathways special, contrary to what the prior techniques require using as markers TK, APRT, XGPRT or DHFR genes.
• the method according to the invention can also be applied to the study of the relative efficiencies of different promoters, whether these efficiencies are expressed in transformation yields with respect to a given type of cell line or with respect to the more or less important number of eukaryotic cells of different origins that can transform each of the promoters tested. It is in this respect interesting to note the very particular effectiveness of the promoters which normally control the transcription of the TK genes.
• the invention therefore provides markers which can be expressed in various mammalian cell lines, particularly in cell lines devoid of any oncogenic character, such as, for example, the heterolooid Vero cell lines of non-human primates, such as those described by PETRICCIANI, JC, KIRSCHSTEIN, RL, HINES, JE, WALLACE, RE and MARTIN, DP (1973), "J. Natl. Cancer Inst.” 51, 191-196, the latter cells having been found to be non-tumorigenic, even when tested in monkeys subjected to immunosuppressive treatment.
• any oncogenic character such as, for example, the heterolooid Vero cell lines of non-human primates, such as those described by PETRICCIANI, JC, KIRSCHSTEIN, RL, HINES, JE, WALLACE, RE and MARTIN, DP (1973), "J. Natl. Cancer Inst.” 51, 191-196, the latter cells having been found to be non-tumori
• non-tumorigenic cells available in banks of cells of the ATCC CCL 81 type (Vero cells) or kidney cells of the vervet monkey or of other non-tumorigenic eukaryotic cells which are preserved in the American Type Culture Collection.
• ATCC CCL 81 type Vero cells
• kidney cells of the vervet monkey or of other non-tumorigenic eukaryotic cells which are preserved in the American Type Culture Collection.
• the labeled cells obtained are therefore particularly useful for the synthesis of products intended for biological or therapeutic uses, whether these are polypeptide sequences corresponding to the HBs antigen of the viral hepatitis B virus, as more particularly envisaged above, or any other active ingredient in vaccines or other useful proteins, such as such as insulin or interferon.
• the invention extends to all the equivalents of the various markers which have been described or defined above. In this regard, it is also important to make the following additional comments.
• eukaryotic promoter known or recognized by its ability to direct the expression in a eukaryotic cell of a gene which is normally associated with it also extends to promoters associated with genes homologous in other types of cells or microorganisms, including promoters under the control of which there are viral genes which code for a protein homologous to that specified by the corresponding eukaryotic gene.
• these are promoters associated with "complementation DNA”, in the sense given to this expression in European patent application No. 80400828 filed on June 9, 1980. In particular, it was makes use of such a promoter in the above example.
• the invention naturally and generally relates to all the methods of labeling eukaryotic cells, characterized by bringing such cells into contact with a marker as defined above, under conditions suitable for allowing the transformation of at least part of these cells and by culturing these cells in a medium suitable for their development and containing an antibiotic inactivable by the enzyme for which codes the DNA sequence of the above marker.
• the invention also relates to eukaryotic cells, in particular originating from monkeys or humans, more particularly suitable for the manufacture of vaccines and containing the marker according to the invention as a selective internal genetic marker.

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