WO2004101819A1 - Procede d'identification de lignees cellulaires - Google Patents

Procede d'identification de lignees cellulaires Download PDF

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Publication number
WO2004101819A1
WO2004101819A1 PCT/EP2004/005095 EP2004005095W WO2004101819A1 WO 2004101819 A1 WO2004101819 A1 WO 2004101819A1 EP 2004005095 W EP2004005095 W EP 2004005095W WO 2004101819 A1 WO2004101819 A1 WO 2004101819A1
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WIPO (PCT)
Prior art keywords
dna
marker dna
marker
sequence
random sequence
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PCT/EP2004/005095
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German (de)
English (en)
Inventor
Gerhard P. PÜSCHEL
Frank NEUSCHÄFER-RUBE
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Universität Potsdam
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Publication date
Application filed by Universität Potsdam filed Critical Universität Potsdam
Priority to EP04732306A priority Critical patent/EP1623043A1/fr
Publication of WO2004101819A1 publication Critical patent/WO2004101819A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the present invention relates to a method for identifying cell lines, in particular animal and human cell lines, which enables rapid and targeted identification of the cell lines to be examined.
  • a specific marker DNA which has at least one random sequence, is introduced into the cell line, and this marker DNA is detected using a suitable method via oligonucleotide hybridization.
  • the marker DNA can also contain further sequences or genes.
  • contaminations with bacteria, yeast, mold, parasites, mycoplasma and viruses there are also contaminations with bacteria, yeast, mold, parasites, mycoplasma and viruses.
  • contamination with bacteria, fungi or viruses can be identified due to a morphological change in the cell line, a changed growth, the discoloration of the culture medium or similar indications.
  • Contamination with other cell lines is problematic and can be morphologically very similar to the original culture. The introduced cells can, for example, proliferate faster and thus overgrow the original culture without this being noticed.
  • Tanabe Tanabe, H., Nakagawa, Y., Minegishi, D., Kurematsu, M., Masui, T. and Mizusawa, H., Tiss. Cult. Res. Commun. , 18: 329-338, 1999.
  • STR short tandem repeat
  • GFP green fluorescence protein
  • a specific marker DNA which has at least one random sequence, is introduced into the cell line, and this marker DNA is detected using a suitable method via oligonucleotide hybridization
  • the term "specific marker DNA” refers to its own, distinctive DNA section which is introduced into the corresponding cell line.
  • the marker DNA can also contain further sequences or genes.
  • a “random sequence” in the sense of the invention is to be understood as a sequence whose base sequence in the genome of the cell naturally occurs in a section of approximately 20 kb is not present upstream and downstream of the integration site of the marker DNA. Optimally, the random sequence does not even exist in the genome of the cell.
  • a cell can contain several random sequences that can be inserted into the cell one after the other or simultaneously in a specific combination.
  • the method for detecting the marker DNA preferably comprises the use of labeled probes or PCR.
  • this essentially means the use of specific primers which are complementary to the random sequence and which provide rapid identification using the polymerase chain reaction (PCR).
  • Further detection methods are known in the prior art and include, among other things, the use of differently labeled probes or the use of newer technical methods which are suitable for detecting a random sequence in accordance with the invention.
  • the marker DNA preferably comprises between 10 and 500 nucleotides.
  • the marker DNA particularly preferably comprises between 15 and 60 nucleotides.
  • the random sequence according to the invention can be identical to the marker DNA or can also represent only a part thereof.
  • At least the random sequence of the introduced marker DNA is unique to the cell genome.
  • successive labeling steps of cells should not be excluded.
  • a combination of at least two marker DNA sequences can be used.
  • a marker DNA can contain several random sequences. Different marker DNAs can be combined in order to enable a specific marking of several successively marked cell lines. This enables derived cell lines to be unequivocally assigned to the original line.
  • the marker DNA is flanked by identical restriction enzyme sites.
  • this marker DNA can also be flanked by various restriction enzyme sites.
  • the cell lines to be identified can be selected from virally infected cells, bacterial cells, plant, fungal, animal and in particular human cells. Mixtures of cells can also be used. Examples of cell lines are lines from E. coli; Lactococcus lactis; Bacillus subtilis, Saccharomyces, Hansenula, Drosophila, Arabidopsis, monkey and human cell lines. Other lines are hybridoma lines and the like.
  • the cell lines to be identified carry foreign genes (also referred to here as "gene of interest") which differ only slightly from one another in their sequence.
  • the cell lines to be identified can also express foreign genes whose proteins differ only slightly from one another in their sequence.
  • a slight sequence difference relates to the exchange of individual nucleotides, smaller sequence segments, individual amino acids or smaller amino acid sequences, which conventionally only enable the DNA sequence of the gene to be expressed to be identified with the aid of DNA sequencing after isolation.
  • the oligonucleotides used to detect the marker DNA are preferably both complementary to the marker DNA.
  • at least one of the oligonucleotides used to detect the marker DNA is complementary to the marker DNA and the other oligonucleotide is complementary to a region outside the marker DNA.
  • this enables the establishment of a standard PCR if a second oligonucleotide sequence is used as the primer for the PCR, which originates from a vector region, and on the other hand the establishment of a user-specific PCR by using a primer which is complementary to the target -DNA is that which should be introduced into the cell line with the aid of the transfection.
  • the marker DNA and / or the random sequence is particularly preferably designed as an inverted repeat or palindrome. This means that the entire sequence of the marker DNA or only a part (such as the random sequence) of the marker DNA is repeated in mirror image.
  • Another aspect of the present invention relates to the use of marker DNA sequences which have a random sequence for identifying cell lines.
  • a further embodiment relates to a transfection construct which comprises at least one marker DNA for introduction into a cell line, the marker DNA having at least one random sequence.
  • His gene of interest only has to be introduced into the transfection construct with the aid of suitable restriction interfaces or with the help of homologous recombination, without that additional cloning of the marker DNA is necessary. In the present case, this is already on the transfection construct and thus enables the inventor to make available a whole range of different transfection vectors that can be used directly by the consumer.
  • kits for identifying cell lines which contains at least the following components: a) a marker DNA which has at least one random sequence and b) an oligonucleotide which has a sequence which is at least partially complementary to the random sequence.
  • the kit contains the following components: a) a vector set, the vectors having different marker DNA sequences each having at least one random sequence and b) oligonucleotides each having at least partially sequences complementary to the random sequences.
  • the phrase "at least partially” refers to the fact that the primers on the one hand bind to the random sequence (or several random sequences) present in the marker DNA, but can also be partially complementary to other areas of the marker DNA.
  • the kit can also contain the instructions for use required to carry out the corresponding identification.
  • 1 shows the identification of four different cell lines by PCR with cell line-specific primers.
  • Fig. 2 shows a simplified process diagram for the subsequent incorporation of a marker DNA into an existing plasmid.
  • Figure 3 shows a possible way of synthesizing polylinker flanking marker DNA.
  • Figure 4 shows another possible way of synthesizing polylinker-flanking marker DNA.
  • Fig. 1 shows the photo of an agarose gel, which was taken after gel electrophoresis.
  • the light bands represent the areas in which DNA molecules, separated according to their molecular weight, have accumulated. They are stained with ethidium bromide and are therefore visible under UV radiation.
  • the investigation was carried out according to the method described in Example 1, the four cell lines being numbered 307 to 310 and the primers being numbered accordingly. Under the corresponding stringent PCR conditions, only the primer that has the exactly complementary random sequence binds to the marker DNA. The four transformed cell lines could thus be identified with a high degree of certainty.
  • the standard additionally plotted on the left lane serves to estimate the size of the DNA identified in the PCR.
  • FIG. 2 schematically shows the process shown in Example 2 as a process diagram.
  • FIGS. 3 and 4 represent process sequences which are explained in more detail in example 4 as the first and third possibility.
  • Animal and human cell cultures can be transfected by various methods. There are basically two types of processes. In addition to viral integration of DNA into the genome of the target cell, the second group introduces DNA into the cell in various ways, which is then randomly or specifically integrated into the genome and with the help of a suitable selection pressure for stable integration of the transfected DNA is selected. In addition to the transfection systems with retroviral or adenoviral genes, an introduction of foreign DNA can also be carried out via calcium phosphate precipitation, with the help of DAE dextran, through liposomes or lipid-mediated methods, microinjection, electroporation, receptor-mediated gene transfer or ballistic Transfection can be achieved. Such cell lines genetically modified in this way can often not be distinguished from one another phenotypically.
  • the present invention provides a method by which it is possible to identify cells transfected with different constructs using a simple PCR method. Analogously, suitable methods can be used to identify transfected cell lines of other origin.
  • a marker DNA is introduced into the construct specifically via restriction sites or homologous recombination, or a set of standard transfection vectors into which the target gene can be cloned is made available to the consumer.
  • a marker DNA is provided at the 3 'end, which among other things. includes a random sequence. This random sequence is unique due to its randomly assembled nucleotide sequence and a corresponding length and can be used under very stringent conditions in the target cell with the help of e.g. PCR can be detected.
  • hybridization in the sense of the present invention is to be understood as binding to form a duplex structure of an oligonucleotide to a completely complementary sequence in the sense of the Watson-Crick base pairings in the sample DNA.
  • very stringent hybridization conditions such conditions are to be avoided. stand in which a hybridization at 60 ° C in 2.5 x SSC buffer, followed by several washing steps at 37 ° C in a lower buffer concentration and remains stable.
  • DNA is extracted from the corresponding cell clones by methods known in the prior art, in order to use them in the PCR in a suitable dilution, or to insert cells directly in the PCR, which by the corresponding denaturation step during the PCR reaction are destroyed and the DNA to be examined is released.
  • the method of the present invention also offers the possibility for non-transgenic cell lines to be identified beyond doubt by the introduction of suitable marker DNA sections.
  • the integration of a small segment of marker DNA enables different cell lines from different organs and organisms to be differentiated.
  • the labeling method of the present invention can provide an exceptionally wide variety of different marker DNA segments and is far superior to the conventional marker genes which require expression of the host cell.
  • animal cell lines are marked as examples.
  • the method is basically equally applicable to all cell lines to be identified.
  • HEK293 cells were transfected with the four vectors. Individual cell lines that had stably integrated the vector construct into the genome were isolated by selection with the antibiotic G418 and single cell cloning. The cell lines established in this way were cultivated under standard conditions.
  • genomic DNA was isolated from the four selected cell lines by ethanol precipitation.
  • the DNA obtained in this way was used in a PCR reaction with marker-specific primers and a primer which was identical for all cell lines and corresponded to a sequence section in the transfected DNA. Under the selected conditions, a PCR product could only be detected in the DNAs of the cell lines, which carried the sequence specific for the marker primer (FIG. 1).
  • Example 2 provides an alternative method.
  • the marker DNA is inserted into the already existing plasmid construct.
  • the marker DNA is restricted with an enzyme which cuts the vector polylinker in the 3 'region of the protein-coding cDNA region of the vector and clones behind the protein-coding region.
  • the marker DNA is joined twice directly behind one another in opposite directions (mirror image, inverted repeat) with a polylink notch region in front of it. This DNA fragment is cut with the desired restriction enzyme and ligated into the dephospholated vector cut with the same enzyme. (Fig. 2)
  • the marker sequence as an inverted repeat, it is possible to establish a standard PCR protocol that is independent of the sequence of the protein-coding region that may be transfected.
  • a PCR is carried out with the marker primer on the one hand and the vector-specific primer located in the 3 'thereof on the other hand (see FIG. 2, standard PCR). This enables the use of the strictest possible PCR conditions and thereby minimizes the amplification of false positive bands. If a protein cDNA-specific primer is used, the user can establish a PCR protocol specific for his cDNA (see FIG. 2, user PCR).
  • a set of vectors is provided, which have a unique marker sequence behind the polylinker.
  • Such a vector set can be used to clone protein-coding DNA regions directly into corresponding interfaces and thereby enables the inventive technique to be used quickly and without problems.
  • Vectors containing a unique marker DNA are prepared by inserting a synthetic, double-stranded oligonucleotide with overhanging ends into the most distant from the transcription start site of the polylinker or another suitable interface in the immediate vicinity of the polylinker, which contains the marker DNA sequence in a mirror image arrangement and can therefore serve as a template for both the user PCR and the standard PCR with the same primer.
  • Marker DNA sequence can be displayed in a polylinker.
  • a DNA fragment is amplified by PCR, a vector with a suitable polylinker being selected as the template.
  • One primer binds to one end of the polylinker.
  • This primer is extended 5 'by the desired marker sequence and 5' by this marker sequence by a sequence coding for a restriction interface not present in the polylinker.
  • the second primer starts inside the vector beyond the polylinker.
  • the PCR product is restricted with the non-polylinker cutting enzyme.
  • the large fragments are cleaned and ligated.
  • the ligation product is cut with the outer enzyme located in the polylinker and ligated into a dephosphorylated vector cut with the same enzyme (FIG. 3).
  • the PCR approach is halved, half is cut with the enzyme not cutting in the polylinker and the outermost enzyme in the polylinker.
  • the second half with the enzyme not cutting in the polylinker and the penultimate enzyme in the polylinker.
  • the two restriction approaches are brought into a ligation with the vector cut with the two enzymes located in the outer polylinker region.
  • a fully synthetic double-stranded DNA fragment containing the inverted repeat as a marker DNA sequence with overhanging ends is cloned into the central interface of a vector to be established, the polylinker of which contains each interface in a mirror image arrangement (FIG. 4 ).
  • the following example relates to a further variant of the method and the method according to the invention.
  • the enzyme X (and / or for example a receptor, such as the prostanoid receptor described, inter alia, in Biochem J. 2003 Apr 15; 371 (Pt 2): 443-9, in which His-81 to Ala mutants in the transmembrane domain 2 and Arg- 291 to Leu mutants in transmembrane domain 7 from the wild type) is of potential interest for a biotechnological process.
  • the enzyme catalyzes an undesirable side reaction that results in a loss of product quality. This side reaction can be suppressed by a single amino acid substitution within or near the active site without affecting the desired main reaction.
  • cDNA constructs were generated by site-directed mutagenesis which code for the same enzyme with only a single amino acid substitution in order to optimize the enzyme for the biotechnological process. All cDNAs were cloned into the same expression vector and cell lines were generated with these cDNAs in an identical cellular background. These cell lines differ in a single or very few known nucleotide exchanges in the transgene.
  • Figure 5 shows the transgene of the wild type and an optimized enzyme. The cell lines can only be identified by SNP analysis or sequencing of the transgene. Switching the cell lines can cause serious damage if you use a cell line that carries the wild-type gene instead of the optimized enzyme.
  • a further palindromic DNA sequence was inserted into the vector together with the cDNA of the enzyme (FIG. 6).
  • This small DNA sequence does not change the properties of the cell line that carries the transgene, nor does it change the properties of the enzyme gene or its expression.
  • the small DNA sequence is specific for each of the cell lines and serves as the target for a specific PCR primer that is in Combination with a vector-specific primer or a transgene-specific primer allows unambiguous identification of the cell line in a single PCR reaction (FIG. 6) of unpurified genomic DNA. This enables the cell line that expresses the optimized enzyme to be identified quickly, conveniently and at low cost in a single PCR reaction at the beginning of each experiment or production cycle. This is an important aspect of quality control, for example.

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Abstract

La présente invention concerne un procédé d'identification de lignées cellulaires, en particulier de lignées cellulaires animales et humaines, lequel permet d'identifier rapidement et de façon ciblée les lignées cellulaires à analyser. Selon ledit procédé, un ADN marqueur spécifique comportant au moins une séquence aléatoire est introduit dans la lignée cellulaire, et cet ADN marqueur est détecté selon un procédé approprié, à savoir par hybridation d'oligonucléotides. Cet ADN marqueur peut, en plus de la séquence aléatoire qu'il doit présenter au minimum ou des séquences aléatoires qu'il présente, comporter au moins d'autres séquences ou gènes.
PCT/EP2004/005095 2003-05-13 2004-05-12 Procede d'identification de lignees cellulaires WO2004101819A1 (fr)

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EP04732306A EP1623043A1 (fr) 2003-05-13 2004-05-12 Procede d'identification de lignees cellulaires

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DE2003121480 DE10321480B4 (de) 2003-05-13 2003-05-13 Verfahren zur Identifizierung von Zellinien
DE10321480.1 2003-05-13

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048822A1 (fr) * 1996-06-17 1997-12-24 Microcide Pharmaceuticals, Inc. Methodes d'analyse au moyen de groupes de souches microbiennes
WO1998055657A1 (fr) * 1997-06-05 1998-12-10 Cellstore Procedes et reactifs permettant d'indexer et de coder des acides nucleiques
WO1999055886A1 (fr) * 1998-04-24 1999-11-04 Genova Pharmaceuticals Corporation Decouverte de genes fonctionnels
DE10027218A1 (de) * 2000-05-31 2001-12-06 Hubert Bernauer Artifizielle genetische Markierung mit synthetischer DNA
WO2002014553A2 (fr) * 2000-08-11 2002-02-21 Favrille, Inc. Systeme d'identification par vecteur moleculaire
GB2376686A (en) * 2001-02-10 2002-12-24 Nat Inst Of Agricultural Botan Storage of encoded information within biological macromolecules

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6153389A (en) * 1999-02-22 2000-11-28 Haarer; Brian K. DNA additives as a mechanism for unambiguously marking biological samples
DE19934573C2 (de) * 1999-07-22 2002-12-05 November Ag Molekulare Medizin Verfahren zur Markierung von festen, flüssigen und gasförmigen Substanzen
US6629038B1 (en) * 2000-09-05 2003-09-30 Yeda Research And Development Co. Ltd. Method and system for identifying commercially distributed organisms

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997048822A1 (fr) * 1996-06-17 1997-12-24 Microcide Pharmaceuticals, Inc. Methodes d'analyse au moyen de groupes de souches microbiennes
WO1998055657A1 (fr) * 1997-06-05 1998-12-10 Cellstore Procedes et reactifs permettant d'indexer et de coder des acides nucleiques
WO1999055886A1 (fr) * 1998-04-24 1999-11-04 Genova Pharmaceuticals Corporation Decouverte de genes fonctionnels
DE10027218A1 (de) * 2000-05-31 2001-12-06 Hubert Bernauer Artifizielle genetische Markierung mit synthetischer DNA
WO2002014553A2 (fr) * 2000-08-11 2002-02-21 Favrille, Inc. Systeme d'identification par vecteur moleculaire
GB2376686A (en) * 2001-02-10 2002-12-24 Nat Inst Of Agricultural Botan Storage of encoded information within biological macromolecules

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHOEMAKER D D ET AL: "QUANTITATIVE PHENOTYPIC ANALYSIS OF YEAST DELETION MUTANTS USING A HIGHLY PARALLEL MOLECULAR BAR-CODING STRATEGY", NATURE GENETICS, NEW YORK, NY, US, vol. 14, no. 4, 1 December 1996 (1996-12-01), pages 450 - 456, XP002043431, ISSN: 1061-4036 *

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EP1623043A1 (fr) 2006-02-08
DE10321480B4 (de) 2005-07-21
DE10321480A1 (de) 2005-03-03

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