Identification of Thymus Specific and Developmentally Regulated Genes by an Improved Version of the mRNA Differential Display Technique

摘要

During embryogenesis in mouse, the thymus is seeded by waves of hematopoietic stem cellsthat provide the first peripheral T lymphocytes after birth. It is known that embryo thymocytesand adult thymocytes have different phenotypic and functional features. The identificationof genes expressed in the thymus only during embryogenesis would help to understandthe molecular basis underlying these characteristics. We used the mRNA differential displaytechnique to compare gene expression between thymus and kidney from embryo (171/2 days)and adult mice. This technique is the method of choice for comparing gene expressionbecause it is able to display rapidly and simultaneously the mRNA complement from severaldifferent types of cells. The major drawback of the method is that it leads to the cloning ofmany false positives and therefore needs a high throughput method to screen for the truly differentiallyexpressed cDNAs. We combined advantages from previously described methodsin order to develop a new version of the mRNA differential display technique that is fast,cheap, and reliable. Instead of oligo dT priming, we used random hexameres for the reversetranscription of total RNA and 10-mer primers for the amplification of internal parts of thecDNAs. We obtained reproducible and clean patterns of discrete bands. We were able to easilyidentify DNAs differentially amplified between embryo and adult tissues (embryo specific;E 58.73), between thymus and kidney (thymus specific; Thy 52.54), or between embryoand adult thymus (embryo thymus specific; E Thy 58.73) cDNA fragments. After reamplification,cloning, and sequencing of these DNA fragments, it appeared that in most cases, oneband corresponded to a single DNA sequence. On a northern blot, each of these candidategenes recognized a transcript that is differentially expressed as expected. Thus, we report anoptimized, reproducible, and fast mRNA differential display method that overcomes the usualproblems met with the originally described technique or its reported modifications.