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

The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg +/- ). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg +/- mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg +/- mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg +/- mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.

• Klíčová slova
• Age-related deterioration of Purkinje cells, Attention deficit hyperactivity disorder, Calcium homeostasis, Cerebellum, Foliation defects, GABA signaling, Hyperactivity, Islet1 transcription factor, Purkinje cells, Transgenic mouse, Vestibular system,
• MeSH
• hyperkineze metabolismus   patologie   MeSH
• mozeček metabolismus   patologie   MeSH
• myši transgenní MeSH
• myši MeSH
• proteiny s homeodoménou LIM biosyntéza   MeSH
• transkripční faktor PAX2 biosyntéza   MeSH
• transkripční faktory biosyntéza   MeSH
• vestibulární aparát metabolismus   patologie   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
• insulin gene enhancer binding protein Isl-1 MeSH Prohlížeč
• Pax2 protein, mouse MeSH Prohlížeč
• proteiny s homeodoménou LIM MeSH
• transkripční faktor PAX2 MeSH
• transkripční faktory MeSH

Sperm-mediated transgenesis of frogs Xenopus laevis with retroviral Rous sarcoma virus DNA is described and the transgenic progeny is characterized. Correlation between the high expression of src gene and defective morphogenesis of frog embryos is discussed.

• MeSH
• DNA virů genetika   MeSH
• fertilizace in vitro MeSH
• geneticky modifikovaná zvířata MeSH
• ovum cytologie   MeSH
• polymerázová řetězová reakce MeSH
• skupina kinas odvozených od src-genu genetika   metabolismus   MeSH
• Southernův blotting MeSH
• spermie cytologie   MeSH
• technika přenosu genů * MeSH
• techniky in vitro MeSH
• transgeny * MeSH
• viry ptačího sarkomu genetika   MeSH
• Xenopus laevis genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA virů MeSH
• skupina kinas odvozených od src-genu MeSH

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

• Klíčová slova
• P1 gene, MicroRNA, Potyvirus, SMV, Transgenic, Virus resistance,
• MeSH
• geneticky modifikované rostliny * genetika   virologie   imunologie   MeSH
• Glycine max genetika   virologie   imunologie   MeSH
• mikro RNA * genetika   MeSH
• nemoci rostlin * virologie   genetika   imunologie   MeSH
• odolnost vůči nemocem * genetika   MeSH
• Potyvirus * patogenita   genetika   MeSH
• RNA interference MeSH
• tabák * genetika   virologie   imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mikro RNA * MeSH

RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.

• MeSH
• myši transgenní MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• RNA interference * MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The aim of this study was to determine whether there are differences in the karyotypes between transgenic and non-transgenic or control rabbits. New Zealand White transgenic rabbits (F1 generation) were obtained after breeding of transgenic founder rabbits that were derived from single--SM--or double microinjection--DM--with a WAP-hFVIII transgene. C-metaphase plates were obtained from short-time culture of peripheral blood lymphocytes synchronized by the addition of colcemide. A significantly higher rate of aneuploidy was observed in c-metaphase spreads of transgenic (56-66%) rabbits, as compared to non-transgenic ones (28-38%) (P < 0.05; P < 0.01). The patterns of chromosome banding were identical in both groups of rabbits. No structural aberrations were revealed in either group. These findings demonstrate that transgenic rabbits have a higher frequency of numerical chromosomal aberrations in their peripheral blood lymphocytes than normal rabbits, but without apparent deleterious effects on health or reproduction.

• MeSH
• aneuploidie * MeSH
• chov MeSH
• chromozomy genetika   MeSH
• diploidie MeSH
• geneticky modifikovaná zvířata genetika   MeSH
• karyotypizace MeSH
• králíci genetika   MeSH
• lymfocyty cytologie   MeSH
• metafáze MeSH
• pruhování chromozomů MeSH
• zvířata MeSH
• Check Tag
• králíci genetika   MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Green Fluorescent protein (GFP) transgenic animals are accepted tools for studying various physiological processes, including organ development and cell migration. However, several in vivo studies claimed that GFP may impair transgenic animals' health. Glomerulosclerosis was observed in transgenic mice and rabbits with ubiquitous reporter protein expression. Heart-specific GFP expression evoked dilated cardiomyopathy and altered cardiac function in transgenic mouse and zebrafish lines, respectively. Moreover, growth retardation and increased axon swelling were observed in GFP and yellow fluorescent protein (YFP) transgenic mice, respectively. This review will focus on the potential drawbacks of the applications of GFP transgenic animals in biomedical research.

• MeSH
• biomedicínský výzkum metody   trendy   MeSH
• dánio pruhované MeSH
• geneticky modifikovaná zvířata genetika   MeSH
• králíci MeSH
• myši transgenní MeSH
• myši MeSH
• vývojová regulace genové exprese MeSH
• zelené fluorescenční proteiny biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• zelené fluorescenční proteiny MeSH

Fourteen genetically modified lines of alfalfa (Medicago sativa) containing the gene Ov from Japanese quail, coding for a methionine-rich protein ovalbumin, were evaluated for nodulation ability and concentration of aerobic bacteria in the rhizosphere. The transgenic lines were derived from a highly regenerable genotype Rg9/I-14-22, selected from cv. Lucia. On selective media, a higher concentration of ammonifying bacteria, bacterial spores, denitrifying and nitrifying bacteria were observed in the rhizosphere of transgenic clonesand, on the other hand, lower concentration of cellulolytic bacteria and Azotobacter spp. compared with the rhizosphere of non-transgenic clone SE/22-GT2. A statistically significant difference in the concentration of all the bacterial types was found between samples taken from two types of substrates (i.e. sterile vs. nonsterile). Higher bacterial concentration (measured as colony forming units per g soil dry mass) were observed for all tested groups of culturable bacteria in the non-sterile substrate. The presence of Azotobacter spp. was found only in the rhizosphere of plants grown in non-sterile soil in which the highest number of fertile soil particles (97 %) was observed in transgenic clones SE/22-9-1-12 and SE/22-11-1-1S.1. Concentration of bacteria involved in the N cycle in the soil was increased in the rhizosphere of transgenic clones and decreased in the rhizosphere of non-transgenic plants compared with the average value. In spite of some differences in colony numbers in samples isolated from the root rhizosphere of transgenic and nontransgenic alfalfa plants, we could not detect any statistically significant difference between individual lines.

• MeSH
• aerobní bakterie klasifikace   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• Azotobacter klasifikace   izolace a purifikace   MeSH
• celulosa metabolismus   MeSH
• geneticky modifikované rostliny mikrobiologie   MeSH
• kořeny rostlin genetika   mikrobiologie   MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• Medicago sativa genetika   mikrobiologie   MeSH
• ovalbumin genetika   MeSH
• půdní mikrobiologie * MeSH
• spory bakteriální izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• celulosa MeSH
• kvartérní amoniové sloučeniny MeSH
• ovalbumin MeSH

WAP is being recognized as the principal milk protein expressed in pregnant or lactating females of several mammalian species. Recently, it has been shown that the 6.3-kb 5' untranslated region of the rWAP gene is able to control, and almost completely restrict, the expression of the transgene into the mammary gland of the transgenic animal. We cloned the genomic fragment carrying the rWAP gene locus from the rabbit phage genomic library and used the 8.5-kb long 5' untranslated part of the rWAP gene to target the expression of hEPO, cloned from the human phage genomic library, into the mammary gland of the mouse. The vectors, carrying either the hEPO gene or the rWAP-hEPO hybrid gene, were injected into the mouse ova, and 12 transgenic animals were identified by PCR and Southern blot from the progeny of 168 tested littermates. Transgenic mice were viable, fertile and displayed a normal development. Recombinant human erythropoietin was produced in the milk of a transgenic mouse female at a secretion level of 5.3 mIU/ml, as detected by ELISA. Despite the low production of the transgenic glycoprotein in the milk we demonstrate that the hybrid gene can be expressed in the mammary gland of the host animal. Thus, WAP-based recombinant vectors, with additional optimizing modifications, can be useful for production of therapeutic proteins in the transgenic mammals.

• MeSH
• erythropoetin krev   genetika   MeSH
• exprese genu MeSH
• lidé MeSH
• mikroinjekce MeSH
• mléčné bílkoviny genetika   MeSH
• mléčné žlázy zvířat fyziologie   MeSH
• myši transgenní MeSH
• myši MeSH
• polymerázová řetězová reakce MeSH
• promotorové oblasti (genetika) genetika   MeSH
• transgeny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• erythropoetin MeSH
• mléčné bílkoviny MeSH
• whey acidic proteins MeSH Prohlížeč

Anomalies in the ultrastructure of chloroplasts, from transgenic ipt tobacco, overproducing endogenous cytokinins (CKs) were studied. Detailed analyses of CKs and their metabolites showed that Pssu-ipt tobacco contained enhanced contents of CKs both in leaves and in isolated chloroplasts. The role of CKs in the formation of anomalous structures is suggested. Pssu-ipt chloroplasts frequently formed the distinct peripheral reticulum with a system of caverns that often involved mitochondria and/or peroxisomes. Large crystalloids, which were found in chloroplasts of Pssu-ipt, occupied up to 16% of chloroplast volume. We suggested that the crystalloids were formed by LHC II aggregates. This was supported by analysis of the fluorescence emission spectra at 77 degrees K, chlorophyll a/b ratio, immunogold staining of the structures, and crystallographic unit size analysis.

• MeSH
• alkyltransferasy a aryltransferasy genetika   metabolismus   MeSH
• chloroplasty genetika   metabolismus   ultrastruktura   MeSH
• cytokininy genetika   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny genetika   MeSH
• tabák cytologie   enzymologie   genetika   metabolismus   MeSH
• transgeny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylate isopentenyltransferase MeSH Prohlížeč
• alkyltransferasy a aryltransferasy MeSH
• cytokininy MeSH