In several species, including Xenopus, mouse and human, two members of cyclin A family were identified. Cyclin A2, which is ubiquitously expressed in dividing cells and plays role in DNA replication, entry into mitosis and spindle assembly, and cyclin A1, whose function is less clear and which is expressed in spermatocytes, leukemia cells and in postmitotic multiciliated cells. Deletion of the gene showed that cyclin A1 is essential for male meiosis, but nonessential for female meiosis. Our results revealed, that the cyclin A1 is not only dispensable in oocytes, we show here that its expression is in fact undesirable in these cells. Our data demonstrate that the APC/C and proteasome in oocytes are unable to target sufficiently cyclin A1 before anaphase, which leads into anaphase arrest and direct inhibition of separase. The cyclin A1-induced cell cycle arrest is oocyte-specific and the presence of cyclin A1 in early embryos has no effect on cell cycle progression or chromosome division. Cyclin A1 is therefore not only an important cell cycle regulator with biased expression in germline, being essential for male and damaging for female meiosis, its persistent expression during anaphase in oocytes shows fundamental differences between APC/C function in oocytes and in early embryos.

• MeSH
• anafáze * MeSH
• cyklin A1 fyziologie   MeSH
• cyklin A2 fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• meióza MeSH
• metafáze MeSH
• mikroinjekce MeSH
• myši MeSH
• oocyty cytologie   MeSH
• proteasomový endopeptidasový komplex fyziologie   MeSH
• segregace chromozomů * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Ccna1 protein, mouse MeSH Prohlížeč
• CCNA2 protein, mouse MeSH Prohlížeč
• cyklin A1 MeSH
• cyklin A2 MeSH
• proteasomový endopeptidasový komplex MeSH

As stated by Korpáš and Tomori (1979), cough is the most important airway protective reflex which provides airway defensive responses to nociceptive stimuli. They recognized that active expiratory efforts, due to the activation of caudal ventral respiratory group (cVRG) expiratory premotoneurons, are the prominent component of coughs. Here, we discuss data suggesting that neurons located in the cVRG have an essential role in the generation of both the inspiratory and expiratory components of the cough reflex. Some lines of evidence indicate that cVRG expiratory neurons, when strongly activated, may subserve the alternation of inspiratory and expiratory cough bursts, possibly owing to the presence of axon collaterals. Of note, experimental findings such as blockade or impairment of glutamatergic transmission to the cVRG neurons lead to the view that neurons located in the cVRG are crucial for the production of the complete cough motor pattern. The involvement of bulbospinal expiratory neurons seems unlikely since their activation affects differentially expiratory and inspiratory muscles, while their blockade does not affect baseline inspiratory activity. Thus, other types of cVRG neurons with their medullary projections should have a role and possibly contribute to the fine tuning of the intensity of inspiratory and expiratory efforts.

• MeSH
• 6-kyano-7-nitrochinoxalin-2,3-dion aplikace a dávkování   MeSH
• antagonisté excitačních aminokyselin aplikace a dávkování   MeSH
• kašel patofyziologie   prevence a kontrola   MeSH
• lidé MeSH
• mechanika dýchání účinky léků   fyziologie   MeSH
• medulla oblongata účinky léků   fyziologie   MeSH
• mikroinjekce metody   MeSH
• nadechnutí účinky léků   fyziologie   MeSH
• nervus phrenicus účinky léků   fyziologie   MeSH
• neurony účinky léků   fyziologie   MeSH
• reflex účinky léků   fyziologie   MeSH
• vydechnutí účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• 6-kyano-7-nitrochinoxalin-2,3-dion MeSH
• antagonisté excitačních aminokyselin MeSH

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

• Klíčová slova
• CRISPR-Cas9, Conditional knockout mouse, Floxed allele, Homology-directed repair, Long single-stranded DNA, Machine learning, Mouse, Oligonucleotide, Reproducibility, Transgenesis,
• MeSH
• alely * MeSH
• blastocysta metabolismus   MeSH
• CRISPR-Cas systémy genetika   MeSH
• faktorová analýza statistická MeSH
• mikroinjekce MeSH
• myši knockoutované MeSH
• protein 2 vázající methyl-CpG genetika   metabolismus   MeSH
• protein Cas9 metabolismus   MeSH
• regresní analýza MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• Mecp2 protein, mouse MeSH Prohlížeč
• protein 2 vázající methyl-CpG MeSH
• protein Cas9 MeSH

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
• Názvy látek
• RNA malá jaderná MeSH

The functional significance of having two nuclei in a cell is unknown. Having two stores of genetic material may be advantageous for cell growth. Nuclear protein import is at a critical juncture in the cell to modify cell growth. This study addressed the potential for differential nuclear protein import in two nuclei of the same cell. Isolated adult rat cardiomyocytes were microinjected with an exogenous fluorescent protein conjugated with nuclear localization amino acid sequences to facilitate nuclear import and its detection. Our results demonstrate the rate of nuclear protein import was not significantly different between the two nuclei in the same cell. These data demonstrate that the two nuclei are functionally similar in a binucleated cardiomyocyte, at least as far as nucleocytoplasmic transport is concerned.

• Klíčová slova
• Binuclear cell, Cardiomyocytes, Nuclear protein import, Nucleocytoplasmic trafficking, Nucleus,
• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• kardiomyocyty metabolismus   MeSH
• mikroinjekce MeSH
• potkani Sprague-Dawley MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

An automatic micro-injector was developed for electrophoretic analysis of a microlitre amount of clinical samples, enabling injection of the sample from a Hamilton syringe. The outlet of the syringe needle is located directly opposite the inlet of the separation capillary at a defined distance of the order of hundreds of μm in the injection space. During the injection, the background electrolyte is forced out by air from this space and a drop of the sample is forced out of the syringe by a micro-pump so that it is caught at the entrance to the capillary. From the drop the sample is injected into the capillary by applying a negative pressure pulse or simply by spontaneous injection. The injection space is then filled with background electrolyte, which washes away excess sample and separation is commenced. The injector was tested in electrophoretic separation of a model sample with equimolar concentrations of 100 μM NH4+, K+, Na+, Mg2+ and Li+ in a short capillary with total/effective length of 16.5/11.5 cm. The repeatability of the migration time and peak area expressed as the RSD value is 2% and 4%, respectively. The practical applicability of the injector was verified on the determination of the antiparasitic pentamidine in 10 μL of rat plasma. Electrophoretic separation of pentamidine was performed in 100 mM of acetic acid/NaOH at pH 4.55, the sample consumption per analysis is 125 nL, the separation time is 45 s and the attained LOQ using contactless conductivity detection is 8 μM.

• Klíčová slova
• Capillary electrophoresis, Pentamidine, Rat plasma, Sample injection, Short capillary,
• MeSH
• amoniové sloučeniny analýza   MeSH
• automatizace MeSH
• draslík analýza   MeSH
• elektroforéza kapilární přístrojové vybavení   MeSH
• hořčík analýza   MeSH
• injekční stříkačky * MeSH
• jehly MeSH
• krysa rodu Rattus MeSH
• lithium analýza   MeSH
• mikroinjekce * MeSH
• sodík analýza   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amoniové sloučeniny MeSH
• draslík MeSH
• hořčík MeSH
• lithium MeSH
• sodík MeSH

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

• Klíčová slova
• Blastocysts, Bovine, In vitro fertilization, Inner cell mass exchange, Sheep,
• MeSH
• chiméra embryologie   MeSH
• ektogeneze * MeSH
• embryoblast cytologie   MeSH
• fertilizace in vitro veterinární   MeSH
• IVM techniky veterinární   MeSH
• jatka MeSH
• klonování organismů veterinární   MeSH
• mikroinjekce veterinární   MeSH
• mikromanipulace veterinární   MeSH
• novorozená zvířata MeSH
• ovce domácí MeSH
• ověření koncepční studie MeSH
• přenos embrya veterinární   MeSH
• skot MeSH
• studie proveditelnosti MeSH
• těhotenství MeSH
• trofoblasty cytologie   MeSH
• vývoj plodu * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Itálie 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
• Názvy látek
• antagonisté excitačních aminokyselin MeSH
• GABA antagonisté MeSH
• kyselina kynurenová MeSH
• mezibuněčné signální peptidy a proteiny MeSH
• muscimol MeSH
• salusin-beta, rat MeSH Prohlížeč

Primordial germ cells (PGCs) are the origin of all germ cells in developing embryos. In the sturgeon embryo, PGCs develop from the vegetal hemisphere, which mainly acts as an extraembryonic source of nutrition. Current methods for studying sturgeon PGCs require either killing the fish or using costly and time-consuming histological procedures. Here, we demonstrate that visualization of sterlet (Acipenser ruthenus>) PGCs in vivo is feasible by simply labeling the vegetal hemisphere with fluorescein isothiocyanate (FITC)-dextran. We injected FITC-dextrans, with molecular weights varying between 10 000 and 2 000 000, into the vegetal pole of 1- to 4-cell stage embryos. At the neurula to tail-bud developmental stages, FITC-positive PGC-like cells appeared ventrally around the developing tail bud in the experimental group that received a high-molecular-weight FITC-dextran. The highest average number of FITC-positive PGC-like cells was observed in embryos injected with FITC-dextran having a molecular weight of 500 000 (FD-500). The pattern of migration of the labeled cells was identical to that of PGCs, clearly indicating that the FITC-positive PGC-like cells were PGCs. Labeled vegetal cells, except for the PGCs, were digested and excreted before the embryos starting feeding. FITC-labeled PGCs were observed in the developing gonads of fish for at least 3 mo after injection. We also found that FD-500 could be used to visualize PGCs in other sturgeon species. To the best of our knowledge, this report is the first to demonstrate in any animal species that PGCs can be visualized in vivo for a long period by the injection of a simple reagent.

• Klíčová slova
• developmental biology, early development, fish reproduction, gonad development, primordial germ cells, sturgeon,
• MeSH
• bičík spermie ultrastruktura   MeSH
• dextrany MeSH
• embryo nesavčí ultrastruktura   MeSH
• embryonální vývoj fyziologie   MeSH
• fluorescein-5-isothiokyanát analogy a deriváty   MeSH
• fluorescenční barviva MeSH
• mikroinjekce MeSH
• molekulová hmotnost MeSH
• pohyb buněk MeSH
• ryby fyziologie   MeSH
• zárodečné buňky ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dextrany MeSH
• fluorescein isothiocyanate dextran MeSH Prohlížeč
• fluorescein-5-isothiokyanát MeSH
• fluorescenční barviva MeSH

Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects. We will describe here a simple TALEN-based mutagenesis protocol suitable for the generation of germline mutations in Bombyx mori and Drosophila melanogaster. The protocol includes assembly of specific TAL modules, in vitro synthesis of TALEN RNAs, egg microinjection and mutation detection using PCR analysis. Our procedure allows a high frequency induction of NHEJ mutations, which often allows the reception of homozygous mutants already in the G1.

• Klíčová slova
• Bombyx mori, Drosophila melanogaster, Engineered nucleases, Genotyping, Golden Gate assembly, pBlue-TAL,
• MeSH
• bourec genetika   MeSH
• deoxyribonukleasy genetika   MeSH
• Drosophila melanogaster genetika   MeSH
• dvouřetězcové zlomy DNA MeSH
• mikroinjekce přístrojové vybavení   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená metody   MeSH
• oprava DNA spojením konců * MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyribonukleasy MeSH