Identification of thymus specific and developmentally regulated genes by an improved version of the mRNA differential display technique.
During embryogenesis in mouse, the thymus is seeded by waves of hematopoietic stem cells that provide the first peripheral T lymphocytes after birth. It is known that embryo thymocytes and adult thymocytes have different phenotypic and functional features. The identification of genes expressed in the thymus only during embryogenesis would help to understand the molecular basis underlying these characteristics. We used the mRNA differential display technique 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 expression because it is able to display rapidly and simultaneously the mRNA complement from several different types of cells. The major drawback of the method is that it leads to the cloning of many false positives and therefore needs a high throughput method to screen for the truly differentially expressed cDNAs. We combined advantages from previously described methods in 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 reverse transcription of total RNA and 10-mer primers for the amplification of internal parts of the cDNAs. We obtained reproducible and clean patterns of discrete bands. We were able to easily identify DNAs differentially amplified between embryo and adult tissues (embryo specific; E 58.73), between thymus and kidney (thymus specific; Thy 52.54), or between embryo and 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, one band corresponded to a single DNA sequence. On a northern blot, each of these candidate genes recognized a transcript that is differentially expressed as expected. Thus, we report an optimized, reproducible, and fast mRNA differential display method that overcomes the usual problems met with the originally described technique or its reported modifications.
Pascolo
S
S
Interfakultäres Institut für Zellbiologie, Abt. Immunologie Auf der Morgenstelle 15, Tübingen, Germany. Steve.PASCOLO@UNI-TUEBINGEN.DE
Tsoukatou
D
D
Mamalaki
C
C
D000818:Animals; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005314:Embryonic and Fetal Development / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D007668:Kidney / Q000502:physiology; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D013950:Thymus Gland / Q000502:physiology*
D018076:DNA, Complementary; D012333:RNA, Messenger