RESUMO
Identification of transgenics still requires PCR and genomic Southern blot hybridization of genomic DNA isolated from tail pieces. Furthermore, identification of transgene-expressing transgenics (hereafter called "expressor") requires mRNA analyses (RT-PCR and Northern blot hybridization) or protein analysis (Western blotting and immunohistochemical staining using specific antibodies). These approaches are often labor-intensive and time-consuming. We developed a technique that simplifies the process of screening expressor transgenics using enhanced green fluorescent protein (EGFP), a noninvasive reporter recently utilized in a variety of organisms, including mice, as a tag. We constructed a MNCE transgene consisting of two expression units, MBP-NCre (termed "MN") and CAG-EGFP (termed "CE"). MN consists of a myelin basic protein (MBP) promoter and NCre gene (Cre gene carrying a nuclear localization signal (NLS) sequence at its 5' end). CE consists of a promoter element, CAG composed of cytomegalovirus (CMV) enhancer and chicken beta-actin promoter, and EGFP cDNA. Of a total of 72 F0 mice obtained after pronuclear injection of MNCE at 1-cell egg stage, 15 were found to express EGFP when the tail, eye, and inner surface of the ear were inspected for EGFP fluorescence under UV illumination at weaning stage. These fluorescent mice were found to possess MNCE and to express NCre mRNA in a brain-specific manner. Mice exhibiting no fluorescence were transgenic or nontransgenic. Mice carrying MNCE, but exhibiting no fluorescence, never expressed NCre mRNA in any organs tested. These findings indicate that (i) direct inspection of the surface of mice for fluorescence under UV illumination enables identification of expressor transgenics without performances of the molecular biological analyses mentioned above, and (ii) systemic promoters such as CAG do not affect the tissue-specificity of a tissue-specific promoter such as MBP promoter, which is located upstream of CAG by approximately 2 kb.
Assuntos
DNA Recombinante/genética , Perfilação da Expressão Gênica/métodos , Genes Reporter/genética , Transgenes/genética , Actinas/genética , Animais , Southern Blotting , Encéfalo/metabolismo , Células Cultivadas , Galinhas/genética , Citomegalovirus/genética , Elementos Facilitadores Genéticos/genética , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/genética , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Recombinação Genética/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
We developed a technique that simplifies the process of confirming homozygous transgenics at preimplantation stages, which are the earliest stages used in test breeding, using enhanced green fluorescent protein as a tag. All the blastocysts obtained by mating with the combination of Tg/Tg male (homozygous for transgene) x +/+ female exhibited fluorescence.
Assuntos
Corantes Fluorescentes/metabolismo , Técnicas de Transferência de Genes , Proteínas Luminescentes/metabolismo , Camundongos Transgênicos , Animais , Blastocisto/metabolismo , Feminino , Proteínas de Fluorescência Verde , Homozigoto , Proteínas Luminescentes/genética , Masculino , Camundongos , Transgenes/genéticaRESUMO
Oxidation reactions of 2,5- and 3,6-di-tert-butyl-3H-azepines (1 and 2) with selenium dioxide (SeO(2)) were performed. The oxidation of 1 with SeO(2) gave 3-tert-butyl-7,7-dimethyl-4-oxo-octa-2,5-dienal 3 in 36% yield, 4-tert-butyl-5-(3,3-dimethyl-2-oxo-butylidene)-1, 5-dihydro-pyrrol-2-one 4 in 13% yield, 2, 6-di-tert-butyl-2-pyridinecarbaldehyde 5 in 12% yield, and 4, 7-di-tert-butyl-2H-azepin-2-one (2-azatropone) 6 in 6% yield, respectively. Oxidation of 2 with SeO(2) gave 2, 2-dimethyl-1-[2-(5-tert-butyl)-pyridyl]propanol 7 in 55% yield, and 3,6-di-tert-butyl-2H-azepine 8 in 5% yield, respectively. We found that selenium dioxide oxidation of 1 affords 4-oxo-octa-2,5-dienal 3 by a new ring cleavage reaction of 1, and we described the first synthesis of 2-azatropone 6 from this oxidation of 1. In the case of 2, pyridylpropanol 7 was obtained as the major product. We now report in detail result of these oxidation reactions, which have led to the synthesis of a novel azatropone derivative.
