The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Wilhelm G. Dirks - One of the best experts on this subject based on the ideXlab platform.
-
STR DNA typing of Human Cell Lines: detection of intra- and interspecies cross-contamination.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Wilhelm G. Dirks, Hans G. DrexlerAbstract:Inter- and intraspecies cross-contaminations (CCs) of Human and animal Cells represent a chronic problem in Cell cultures leading to false data. Microsatellite loci in the Human genome harboring short tandem repeat (STR) DNA markers allow individualization of Cell Lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of Human Cell Lines and represents an international reference technique. The major Cell banks of the USA, Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a Human Cell line, the suspected identity can be proven by online verification of customer-made STR data sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent Cells within a Human Cell culture population. Since authentic Cell Lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying identity and purity of Human Cell Lines of interest.
-
High-throughput SNP-based authentication of Human Cell Lines.
International journal of cancer, 2012Co-Authors: Felipe A. Castro, Wilhelm G. Dirks, Silke Fähnrich, Agnes Hotz-wagenblatt, Michael Pawlita, Markus SchmittAbstract:Use of false Cell Lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for Cell line authentication. However, mismatch repair (MMR)-deficient Cell Lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side Lines, and, thus, are likely to be misclassified by STR profiling. On the basis of the high-throughput Luminex platform, we developed a 24-plex single nucleotide polymorphism profiling assay, called multiplex Cell authentication (MCA), for determining authentication of Human Cell Lines. MCA was evaluated by analyzing a collection of 436 Human Cell Lines from the German Collection of Microorganisms and Cell Cultures, previously characterized by eight-loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10(-8) for STR profiling and ~1 × 10(-9) for MCA). MCA enabled the detection of less than 3% of contaminating Human Cells. By analyzing MMR-deficient Cell Lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine Cell line authentication and replace the standard authentication STR technique in case of MSI Cell Lines.
-
Recommendation of short tandem repeat profiling for authenticating Human Cell Lines, stem Cells, and tissues
In vitro cellular & developmental biology. Animal, 2010Co-Authors: Rita Barallon, Wilhelm G. Dirks, Steven R. Bauer, John M. Butler, Amanda Capes-davis, Elmore E, Manohar R. Furtado, Margaret C. Kline, Arihiro Kohara, Georgyi V. LosAbstract:Cell misidentification and cross-contamination have plagued biomedical research for as long as Cells have been employed as research tools. Examples of misidentified Cell Lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous Cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of Cells and Cell Lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of Human Cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating Human Cell Lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating Human Cell Lines, stem Cells, and tissues.
-
Authentication of Scientific Human Cell Lines
Methods in molecular biology (Clifton N.J.), 2005Co-Authors: Wilhelm G. Dirks, Hans G. DrexlerAbstract:Human Cell Lines are an important resource for research and most often used in reverse genetic approaches or as in vitro model systems of Human diseases. In this regard, it is crucial that the Cells faithfully correspond to the purported objects of study. A number of recent publications have shown an unacceptable level of Cell Lines to be false, in part as a result of the nonavailability of a simple and easy DNA profiling technique. We have validated different single- and multiple-locus variable numbers of tandem repeats (VNTRs) enabling the establishment of a noncommercial, but good laboratory practice, method for authentication of Cell Lines by DNA fingerprinting. Polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of six prominent and highly polymorphic minisatellite VNTR loci, requiring only a thermal cycler and an electrophoretic system, was proven as the most reliable tool. Furthermore, the generated banding pattern and the determination of gender allows for verifying the authenticity of a given Human Cell line by simple agarose gel electrophoresis. The combination of rapidly generated DNA profiles based on single-locus VNTR loci and information on banding patterns of Cell Lines of interest by official Cell banks (detailed information at the website www.dsmz.de) constitute a low-cost but highly reliable and robust method, enabling every researcher using Human Cell Lines to easily verify Cell line identity.
-
Short tandem repeat profiling provides an international reference standard for Human Cell Lines
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: John R. W. Masters, Wilhelm G. Dirks, Tadao Ohno, James A. Thomson, Bernadette Daly-burns, Yvonne Reid, Phil Packer, Lorraine Toji, Hideyuki Tanabe, Colin F. ArlettAbstract:Cross-contamination between Cell Lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of Cell Lines are of a different origin or species to that claimed. To test a standard method of Cell line authentication, 253 Human Cell Lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every Cell line. If DNA profiling of Cell Lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.
Annette Mankertz - One of the best experts on this subject based on the ideXlab platform.
-
Infection studies on Human Cell Lines with porcine circovirus type 1 and porcine circovirus type 2.
Xenotransplantation, 2004Co-Authors: Kim Hattermann, Claudia Roedner, Cornelia Schmitt, Tim Finsterbusch, Tobias Steinfeldt, Annette MankertzAbstract:The lack of Human donor organs in allotransplantation has led to a proposal for the use of porcine tissues and organs as alternative therapeutic material for Humans. Besides immunological problems like graft rejection, one of the major concerns is the transmission of porcine microorganisms as viruses, bacteria and fungi to a Human recipient. Human Cell Lines have been infected with porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) to investigate whether PCV can infect and replicate in Human epithelial Cells and lymphocytes. Infection of PCV1 was observed with 293, Hela and Chang liver Cells, infection with PCV2 only in Rd Cells. In addition, religated viral DNA of PCV1 and PCV2 has been used to transfect adherent Human Cell Lines. PCV1 persisted in most Cell Lines without causing any visible changes, while PCV2-transfected Cells showed a cytopathogenic effect. Presence of PCV DNA was detected in Cells and supernatant by PCR, expression of viral proteins by an indirect immune fluorescence assay. A replication assay showed that the replication of PCV DNA was initiated at the origin of replication. When virus-free Cells were inoculated with the supernatant of PCV-infected Human Cells, the infection was not passed. Although PCV gene expression and replication took place in Human Cells, the infection is non-productive. Alteration of protein localization suggests that protein targeting may be disturbed in Human Cells. Copyright Blackwell Munksgaard, 2004
Hans G. Drexler - One of the best experts on this subject based on the ideXlab platform.
-
Prevalence and characterization of murine leukemia virus contamination in Human Cell Lines.
PloS one, 2015Co-Authors: Cord C. Uphoff, Sandra Lange, Sabine A. Denkmann, Henk S. P. Garritsen, Hans G. DrexlerAbstract:Contaminations of Cell cultures with microbiological organisms are well documented and can be managed in Cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in Cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 Human Cell Lines for the presence of murine leukemia viruses (MLV). Nineteen Cell Lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 Cell Lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated Cell Lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary Human Cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected Cell Lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected Cell cultures were infectious to uninfected Cell cultures. In the course of the study we found that contamination of Human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the Human tumor Cells into immune-deficient mice to determine the tumorigenicity of the Cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer Cells during the establishment of Human Cell Lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of Cell cultures for MLV contamination is recommended given a contamination rate of 3.3%.
-
STR DNA typing of Human Cell Lines: detection of intra- and interspecies cross-contamination.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Wilhelm G. Dirks, Hans G. DrexlerAbstract:Inter- and intraspecies cross-contaminations (CCs) of Human and animal Cells represent a chronic problem in Cell cultures leading to false data. Microsatellite loci in the Human genome harboring short tandem repeat (STR) DNA markers allow individualization of Cell Lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of Human Cell Lines and represents an international reference technique. The major Cell banks of the USA, Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a Human Cell line, the suspected identity can be proven by online verification of customer-made STR data sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent Cells within a Human Cell culture population. Since authentic Cell Lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying identity and purity of Human Cell Lines of interest.
-
Authentication of Scientific Human Cell Lines
Methods in molecular biology (Clifton N.J.), 2005Co-Authors: Wilhelm G. Dirks, Hans G. DrexlerAbstract:Human Cell Lines are an important resource for research and most often used in reverse genetic approaches or as in vitro model systems of Human diseases. In this regard, it is crucial that the Cells faithfully correspond to the purported objects of study. A number of recent publications have shown an unacceptable level of Cell Lines to be false, in part as a result of the nonavailability of a simple and easy DNA profiling technique. We have validated different single- and multiple-locus variable numbers of tandem repeats (VNTRs) enabling the establishment of a noncommercial, but good laboratory practice, method for authentication of Cell Lines by DNA fingerprinting. Polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of six prominent and highly polymorphic minisatellite VNTR loci, requiring only a thermal cycler and an electrophoretic system, was proven as the most reliable tool. Furthermore, the generated banding pattern and the determination of gender allows for verifying the authenticity of a given Human Cell line by simple agarose gel electrophoresis. The combination of rapidly generated DNA profiles based on single-locus VNTR loci and information on banding patterns of Cell Lines of interest by official Cell banks (detailed information at the website www.dsmz.de) constitute a low-cost but highly reliable and robust method, enabling every researcher using Human Cell Lines to easily verify Cell line identity.
Kim Hattermann - One of the best experts on this subject based on the ideXlab platform.
-
Infection studies on Human Cell Lines with porcine circovirus type 1 and porcine circovirus type 2.
Xenotransplantation, 2004Co-Authors: Kim Hattermann, Claudia Roedner, Cornelia Schmitt, Tim Finsterbusch, Tobias Steinfeldt, Annette MankertzAbstract:The lack of Human donor organs in allotransplantation has led to a proposal for the use of porcine tissues and organs as alternative therapeutic material for Humans. Besides immunological problems like graft rejection, one of the major concerns is the transmission of porcine microorganisms as viruses, bacteria and fungi to a Human recipient. Human Cell Lines have been infected with porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) to investigate whether PCV can infect and replicate in Human epithelial Cells and lymphocytes. Infection of PCV1 was observed with 293, Hela and Chang liver Cells, infection with PCV2 only in Rd Cells. In addition, religated viral DNA of PCV1 and PCV2 has been used to transfect adherent Human Cell Lines. PCV1 persisted in most Cell Lines without causing any visible changes, while PCV2-transfected Cells showed a cytopathogenic effect. Presence of PCV DNA was detected in Cells and supernatant by PCR, expression of viral proteins by an indirect immune fluorescence assay. A replication assay showed that the replication of PCV DNA was initiated at the origin of replication. When virus-free Cells were inoculated with the supernatant of PCV-infected Human Cells, the infection was not passed. Although PCV gene expression and replication took place in Human Cells, the infection is non-productive. Alteration of protein localization suggests that protein targeting may be disturbed in Human Cells. Copyright Blackwell Munksgaard, 2004
Hamid M Said - One of the best experts on this subject based on the ideXlab platform.
-
Targeting and intraCellular trafficking of clinically relevant hTHTR1 mutations in Human Cell Lines.
Clinical Science, 2007Co-Authors: Veedamali S. Subramanian, Jonathan S Marchant, Hamid M SaidAbstract:The micronutrient thiamine is required for normal growth and development of Human tissues, and is accumulated into Cells through the activity of plasma membrane thiamine transporters, e.g. hTHTR1 (Human thiamine transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in Human Cell Lines derived from epithelia relevant to thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Δ383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased thiamine transport activity underpinned by changes in hTHTR1 expression levels, Cellular targeting and/or protein transport activity.
-
Targeting and intraCellular trafficking of clinically relevant hTHTR1 mutations in Human Cell Lines.
Clinical science (London England : 1979), 2007Co-Authors: Veedamali S. Subramanian, Jonathan S Marchant, Hamid M SaidAbstract:The micronutrient thiamine is required for normal growth and development of Human tissues, and is accumulated into Cells through the activity of plasma membrane thiamine transporters, e.g. hTHTR1 (Human thiamine transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in Human Cell Lines derived from epithelia relevant to thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Delta383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased thiamine transport activity underpinned by changes in hTHTR1 expression levels, Cellular targeting and/or protein transport activity.