Applicability of nonsyngeneic cell models for screening of genes in monogenetic diseases via differential display technique.
Conventional subtraction library techniques or DNA-transfection studies are standard techniques applied for identification and isolation of genes relevant in monogenetic diseases like Fanconi anemia (FA). The differential display technique (DDT) was developed to compare mRNA expression between a mutant cell line and its syngeneic control and allows comparison of almost all mRNA species within a short time. However, for identification of genes relevant in monogenetic diseases, no syngeneic cell model is available. In this report, we show that the use of nonsyngeneic diploid human fibroblasts does not increase the number of differentially displayed bands due to diversity of untranslated regions. cDNA bands with a length of up to 1000 bp were obtained and applied to DDT. After screening of about 13000 cDNA bands, only 0.5% were found to be differentially expressed between FA and control cells. Finally, three mRNAs were cloned and verified in Northern blot experiments to be differentially expressed in FA fibroblasts. The low number of differentially displayed cDNA bands in DDT indicates the usefulness of this statistical, molecular approach for identification of multiple genes dysregulated in gene regulation cascades potentially relevant for cell cycle disturbances.
Planitzer
S A
SA
Boehringer Mannheim Research Center, Penzberg, Germany. simone_planitzer@bmg.boehringer-mannheim.com
Machl
A W
AW
Kubbies
M
M
D001973:Bromodeoxyuridine; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division; D002460:Cell Line; D003626:Data Display; D004171:Diploidy; D005199:Fanconi Anemia / Q000235:genetics*; D005347:Fibroblasts / Q000502:physiology; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D008957:Models, Genetic
D001973:Bromodeoxyuridine