Paraffin histology is one of the most important and commonly used laboratory techniques enabling the study of the microscopic structure of animal and plant tissues. This technique uses paraffin wax, which in liquid form impregnates fixed and dehydrated tissues and allows the preparation of thin sections when solidified in blocks. This protocol on good practices in paraffin histology of Nothobranchius furzeri (Nothobranchiidae) summarizes the authors' current experience in terms of technique, evaluation, and interpretation of sectioned tissues. The steps that precede paraffin block preparation are also presented as they play a key role in maximizing the quality of examined sections. The paraffin technique as described only requires basic laboratory conditions to produce good-quality results. The description of staining methods is limited to Mayer's hematoxylin and eosin (H&E), the routinely used histological dye staining cell nuclei in blue-black (hematein) and cell cytoplasm and connective tissue fibers in shades of pink-red (eosin). Killifish specialists are encouraged to engage in the study of histology and histopathology, taking advantage of interdisciplinary cooperation.

• MeSH
• barvení a značení MeSH
• eosin MeSH
• Fundulidae * MeSH
• mikrotomie MeSH
• parafín MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eosin MeSH
• parafín MeSH

The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and discussion of the benefits and drawbacks of using long double-stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression.

• MeSH
• embryo savčí cytologie   MeSH
• genetické techniky * MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• RNA interference * MeSH
• transgeny MeSH
• umlčování genů MeSH
• vývojová biologie metody   MeSH
• zvířata MeSH
• Check Tag
• myši 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 many model systems. The protocol presented here describes the cloning of a plasmid construct for use in transgenic RNAi experiments in mouse oocytes. The protocol is intended for production of a transgene by cloning an inverted repeat (IR) into the Zp3 transgenic cassette. The procedure begins with the selection of sequences and formulation of the cloning strategy. Subsequently, the IR is cloned, inserted into the transgenic cassette, and characterized by sequencing. Finally, the transgene is released from the cassette, purified, and provided to the transgenic facility.

• MeSH
• Bacteria metabolismus   MeSH
• klonování DNA * MeSH
• molekulární sekvence - údaje MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty cytologie   MeSH
• plazmidy metabolismus   MeSH
• repetitivní sekvence nukleových kyselin MeSH
• RNA interference * MeSH
• sekvence nukleotidů MeSH
• transgeny * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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
• Názvy látek
• dvouvláknová RNA MeSH
• RNA MeSH

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. The production of a transgene for transgenic RNAi in mouse can be accomplished by cloning an inverted repeat (IR) into the Zp3 transgenic cassette. Here, we describe three different strategies that have been used successfully to clone an IR: cloning by ligating polymerase chain reaction (PCR) products, sequential cloning using a short spacer, and sequential cloning using a temporary long spacer. Once cloning has been completed, sequencing the IR is the best way to assure that both arms are sufficiently long and intact; thus, we also describe typical problems one may encounter when sequencing IRs. There are two possible strategies for sequencing a cloned IR: (1) one internal primer at the end of the IR can be used to sequence both arms in a single sequencing run or (2) external primers can be used to sequence both arms separately. Although we have successfully sequenced IRs using both strategies, we suggest using the latter to sequence transgenic constructs.

• MeSH
• genetické techniky * MeSH
• intergenová DNA * MeSH
• klonování DNA metody   MeSH
• lidé MeSH
• modely genetické MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• polymerázová řetězová reakce MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• RNA interference * MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA metody   MeSH
• transgeny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• intergenová DNA * MeSH

RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for a number of model systems. Here, we describe selection of the target sequence for efficient RNAi knockdown in mouse.

• MeSH
• 3' nepřekládaná oblast MeSH
• databáze genetické MeSH
• dvouvláknová RNA metabolismus   MeSH
• exprimované sekvenční adresy MeSH
• genetické techniky * MeSH
• komplementární DNA metabolismus   MeSH
• malá interferující RNA metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• myši MeSH
• posttranskripční úpravy RNA MeSH
• RNA interference * MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• dvouvláknová RNA MeSH
• komplementární DNA MeSH
• malá interferující RNA MeSH
• messenger RNA MeSH