Expression of the recombinase‐activating gene (RAG‐1) in murine early embryogenesis

Abstract

Summary The recombinase activation genes, RAG‐1 and RAG‐2, are expressed together in immature T or B lymphocytes and possess activity to induce V(D)J rearrangement in T cell receptor (TCR) and Ig genes. In vertebrates, only Ig and TCR molecules are reported to have recombination in their development using multiple V, D, J component gene segments. Thus, expression of RAG genes are localized only in lymphoid organs and sites of extrathymic T cell differentiation. In this study, we have used RAG‐1 and RAG‐2 genes as markers of possible genetic recombination in developing murine preimplanation embryos, using the highly sensitive reverse transcriptase polymerase chain reaction (RT‐PCR) technique and in situ hybridization. From 40 preimplantation embryos of various developmental stages we extracted RNA. reverse‐transcribed it into cDNA and used it in RT‐PCR studies. A PCR of 35 cycles disclosed expression of RAG‐1 but not RAG‐2 in morulae and blastocysts. Southern blot hybridization using a specific synthetic oligonucleotide probe for RAG‐1 and RT‐PCR with another primer pair identified RAG‐1 expression in developing embryos. In situ hybridization using a cooled CCD camera also revealed localization of RAG‐1 mRNA in blastocysts. We propose possible genetic recombination during late preimplantation murine embryogenesis which may contribute to the loss of totipotency and differentiation of inner cell mass and trophoectoderm.

Quantitation of Thyroid Peroxidase mRNA in Peripheral Blood for Early Detection of Thyroid Papillary Carcinoma

“…PCR entailed initial denaturation at 94°C for 12 minutes, followed by 50 cycles of 94°C for 1 minute, 58°C for 2 minutes, 72°C for 3 minutes, with final extension at 72°C for 7 minutes. RT-PCR cycles for Tg and ␤ actin were performed as described (7,9). RT-PCR products (10 L) were separated by electrophoresis on a 2.0% agarose gel.…”

Pregnancy and gamma/delta T cells: Taking on the hard questions

“…In humans, an intriguing observation is that the CD56 bright+ /CD16 - cells, numerous at early pregnancy, drastically drop at the second and third trimester and are practically absent at term. Moreover, they express c-kit and the recombinase activating genes (RAG) 1 and RAG2, suggesting TCR rearrangement processes or/and a progenitor nature of these cells [13,17,18]. …”

“…In previous reports, Hayakawa et al [17] in the human system and Kimura et al [18] in the murine system have shown expression of mRNA for RAG-1 and RAG-2 proteins, which are required for TCR rearrangement, in human CD56 bright /CD16 - cells and in murine decidual mononuclear cells respectively. We have confirmed and extended these results showing that transcripts of RAG can be easily detected in purified CD56 + , CD2 + , c-kit + or IL-7R + decidual cells implying an ongoing process of TCR gene rearrangement [13].…”

iRAGu: A Novel Inducible and Reversible Mouse Model for Ubiquitous Recombinase Activity

“…The recombinase complex is tightly regulated at tissue, cellular and molecular levels. First, RAG1 and 2 are both expressed only in lymphocytes, though one or the other are expressed at low levels in different non-lymphoid tissues (19–21). Second, RAG expression is downregulated throughout the G1/S phase of the cell cycle and during the cell divisions following the recombination of an antigen receptor locus (22, 23), which occur primarily after the production of a functional heavy chain of antigen receptor (TCRβ, TCRδ, and IgH loci) and secondly after the expression and the pairing of the light chain to the heavy chain (TCRα, TCRγ, and Igλ or Igκ, respectively) to form the cell surface receptor.…”