Microinjection
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Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.
- MeSH
- HeLa buňky MeSH
- lidé MeSH
- mikroinjekce metody MeSH
- RNA malá jaderná metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- krysa rodu rattus MeSH
- neurony MeSH
- nucleus caudatus MeSH
- serotonin MeSH
- spánek MeSH
- Check Tag
- krysa rodu rattus MeSH
Salusin-beta is newly identified bioactive peptide of 20 amino acids, which is widely distributed in hematopoietic system, endocrine system, and the central nervous system (CNS). Although salusin-beta extensively expressed in the CNS, the central cardiovascular functions of salusin-beta are unclear. Our main objective was to determine the cardiovascular effect of microinjection of salusin-beta into the nucleus tractus solitarii (NTS) in anesthetized rats. Bilateral or unilateral microinjection of salusin-beta (0.94-94 microg/rat) into the NTS dose-dependently decreased blood pressure and heart rate. Bilateral NTS microinjection of salusin-beta (9.4 microg/rat) did not alter baroreflex sensitivity. Prior application of the glutamate receptor antagonist kynurenic acid (0.19 microg/rat, n=9) into the NTS did not alter the salusin-beta (9.4 microg/rat) induced hypotension and bradycardia. However, pretreatment with the GABA receptor agonist muscimol (0.5 ng/rat) within the rostral ventrolateral medulla (RVLM) completely abolished the hypotension (-14+/-5 vs. -3+/-5 mm Hg, P<0.05) and bradycardia (-22+/-6 vs. -6+/-5 bpm, P<0.05) evoked by intra-NTS salusin-beta (9.4 microg/rat). In addition, we found that vagotomy didn't influence the actions of salusin-beta (9.4 microg/rat) in the NTS. In conclusion, our present study shows that microinjection of salusin-beta into the NTS significantly produces hypotension and bradycardia, presumably by suppressing the activities of presympathetic neurons in the RVLM.
- MeSH
- antagonisté excitačních aminokyselin farmakologie MeSH
- baroreflex účinky léků MeSH
- bradykardie chemicky indukované patofyziologie MeSH
- GABA antagonisté farmakologie MeSH
- hemodynamika účinky léků MeSH
- hypotenze chemicky indukované patofyziologie MeSH
- krysa rodu rattus MeSH
- kyselina kynurenová farmakologie MeSH
- medulla oblongata cytologie účinky léků MeSH
- mezibuněčné signální peptidy a proteiny aplikace a dávkování farmakologie MeSH
- mikroinjekce MeSH
- muscimol farmakologie MeSH
- neurony účinky léků MeSH
- nucleus solitarius účinky léků MeSH
- potkani Sprague-Dawley MeSH
- sympatický nervový systém účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. The protocol presented here is designed for the preparation of microgram amounts of double-stranded RNA (dsRNA) for microinjection experiments in mouse oocytes and early embryos. Briefly, dsRNA is produced after annealing sense and antisense RNA strands, or spontaneously during in vitro transcription of an inverted repeat. We usually include RNase T1 treatment of the annealed RNA prior to the purification step in order to remove unannealed single-stranded RNA (ssRNA), which can interfere with the quantification of dsRNA in a nondenaturing agarose gel.
- MeSH
- dvouvláknová RNA genetika MeSH
- genetická transkripce MeSH
- genetické techniky MeSH
- mikroinjekce metody MeSH
- modely genetické MeSH
- molekulární biologie metody MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- oocyty metabolismus MeSH
- plazmidy metabolismus MeSH
- polymerázová řetězová reakce metody MeSH
- RNA interference MeSH
- RNA metabolismus MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase, together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer (SCNT), and it offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing and the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in 1 d; generation of germline-transgenic lines by using this protocol takes ∼1 year.
- MeSH
- genetické vektory MeSH
- geneticky modifikovaná zvířata * MeSH
- genom MeSH
- mikroinjekce MeSH
- prasata genetika MeSH
- technika přenosu genů * MeSH
- transposasy MeSH
- transpozibilní elementy DNA * MeSH
- zárodečné buňky MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
