RESUMO
The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 µM Io for 4 min followed by 5 µM DhL concentration and after 3-h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6-dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI-embryos or SCNT-embryos is reported here for first time.
Assuntos
Bovinos/embriologia , Embrião de Mamíferos/efeitos dos fármacos , Ionomicina/farmacologia , Lactonas/farmacologia , Óvulo/efeitos dos fármacos , Sesquiterpenos/farmacologia , Animais , Clonagem de Organismos , Desenvolvimento Embrionário/efeitos dos fármacos , Lactonas/química , Estrutura Molecular , Técnicas de Transferência Nuclear/veterinária , Partenogênese , Sesquiterpenos/química , Injeções de Esperma Intracitoplásmicas/veterináriaRESUMO
Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23-60%), but with a high frequency of mosaic expression (range 60-85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.
Assuntos
Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Clonagem de Organismos/veterinária , Técnicas de Transferência de Genes/veterinária , Animais , Gatos , Bovinos , Clonagem de Organismos/métodos , Desenvolvimento Embrionário , Feminino , Proteínas de Fluorescência Verde/genética , Cavalos , Masculino , Gravidez , Proteínas Recombinantes/genética , Ovinos , Especificidade da Espécie , Injeções de Esperma Intracitoplásmicas/métodos , Injeções de Esperma Intracitoplásmicas/veterinária , SuínosRESUMO
Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest.
Assuntos
Bovinos/embriologia , Suínos/embriologia , Transposases/genética , Zigoto/metabolismo , Animais , Animais Geneticamente Modificados , Citoplasma , Reação em Cadeia da PolimeraseRESUMO
The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.
Assuntos
Sistemas CRISPR-Cas , Bovinos/embriologia , Fertilização in vitro/veterinária , Engenharia Genética/veterinária , Proteínas Priônicas/metabolismo , Animais , Bovinos/genética , Feto/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Proteínas Priônicas/genéticaRESUMO
Sterols and gibberellins are the main terpenoids in the Ascomycete Fusarium fujikuroi. Their respective precursors squalene and ent-kaur-16-ene (henceforth called kaurene) were the main terpenoids synthesised from radioactive mevalonate by extracts of F. fujikuroi in vitro. Kaurene predominated when the extracts were obtained from mycelia engaged in gibberellin production. Squalene predominated in all other cases, and particularly when the extracts were obtained from mutants with various defects in gibberellin synthesis or nitrogen-fed wild-type cultures. New protein synthesis was required to maintain the production of gibberellins in vivo and of kaurene in vitro, but not to maintain the capacity to produce squalene in vitro. Addition of a nitrogen source to cultures engaged in gibberellin production caused a large, transient increase in the mycelial concentration of L-glutamine and abolished the accumulation of gibberellins immediately and the capacity to produce kaurene later.
Assuntos
Diterpenos do Tipo Caurano , Fusarium/metabolismo , Esqualeno/metabolismo , Sistema Livre de Células , Diterpenos , Fusarium/citologiaRESUMO
A case of colonic endometriosis in a 32 year-old woman in presented. We comment the clinical presentation, with rectal hemorrhages as the only symptom; the involvement of the colonic mucosa in the form of a polypoid lesion and the finding of endometrium in the mucosal biopsy. These exceptional coincidences made the pathological diagnosis possible in the tissue obtained by the endoscopic biopsy.
Assuntos
Endometriose/patologia , Pólipos Intestinais/patologia , Neoplasias Primárias Múltiplas/patologia , Neoplasias do Colo Sigmoide/patologia , Adulto , Biópsia/métodos , Feminino , HumanosRESUMO
We describe a case of hepatic carcinoid tumor that was diagnosed after right hepatectomy. The tumor was initially believed to be metastases from a rectal adenocarcinoma for which surgery had been performed seven years earlier. We highlight the localization of the tumor in the liver only, its large size and rapid growth, as well as the absence of carcinoid syndrome. Diagnostic studies of function and localization are presented. The absence of neoplasia in other locations suggested that this neoplasm was probably a primary hepatic carcinoid tumor. The association of carcinoid tumors with other neoplasms is well-known. However, because of our patient's history and the absence of symptoms, the preoperative diagnosis was incorrect. The definitive diagnosis was confirmed by pathological analysis, allowing specific studies of function and localization to be performed and therapeutic and follow-up measures to be adopted. Although surgery plays a crucial role in the treatment of these tumors, it should be performed in the context of the multidisciplinary management recommended for this disease.
Assuntos
Adenocarcinoma , Tumor Carcinoide/diagnóstico , Erros de Diagnóstico , Neoplasias Hepáticas/diagnóstico , Segunda Neoplasia Primária , Neoplasias Retais , Adenocarcinoma/secundário , Adenocarcinoma/cirurgia , Tumor Carcinoide/patologia , Tumor Carcinoide/cirurgia , Hepatectomia/métodos , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neoplasias Retais/cirurgia , Tomografia Computadorizada por Raios XRESUMO
The inverted papilloma of the urethra is an exceptional entity. It occurs as a smooth surface, sessile tumour located in the prostatic urethra. Microscopically, it is lined with transitional epithelium which is projected inside the tumour, creating strands of cells with normal characteristics. All cases reported up to now have occurred in men aged between 49 and 79. None of the cases had additional urothelial injuries associated. Contribution of a new case of inverted papilloma of the prostatic urethra in a 52-year old male. Transurethral resection of the tumour was the procedure chosen. Evolution was good and no relapse nor further urothelial injuries were observed at the controls carried out over an 8-year follow-up period.
Assuntos
Papiloma/patologia , Neoplasias Uretrais/patologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Malignant tumours of the urachus are very rare and they give certain difficulty in their differential diagnosis with vesical cupula adenocarcinoma. We present a case of mucinous adenocarcinoma of the urachus in a 75-year-old patient and we analyse the behaviour, clinical background, diagnosis and treatment of these tumours.
Assuntos
Adenocarcinoma Mucinoso/diagnóstico , Úraco , Adenocarcinoma Mucinoso/patologia , Adenocarcinoma Mucinoso/cirurgia , Idoso , Diagnóstico Diferencial , Humanos , Masculino , Invasividade Neoplásica , Úraco/patologia , Úraco/cirurgia , Neoplasias da Bexiga Urinária/diagnósticoRESUMO
Embryo disaggregation allows the production of two to four identical offspring from a single cow embryo. In addition, embryo complementation has become the technique of choice to demonstrate the totipotency of embryonic stem cells and induced pluripotent stem cells. Therefore, the aim of this study was to generate a new and simple method by aggregation in the well-of-the-well system to direct each single enhanced green fluorescent protein (egfp) eight-cell blastomere derived from bovine in vitro fertilization embryos to the inner cell mass (ICM) of chimeras produced with fused and asynchronic embryos. To this end, the best conditions to generate in vitro fertilization-fused embryos were determined. Then, the fused (F) and nonfused (NF) embryos were aggregated in two distinct conditions: synchronically (S), with both transgenic and F embryos produced on the same day, and asynchronically (AS), with transgenic embryos produced one day before F embryos. The highest fusion and blastocysts rates were obtained with two pulses of 40 V. The 2ASF and 2ASNF groups showed the best number of blastocysts expressing the EGFP protein (48% and 41%, respectively). Furthermore, the 2ASF group induced the highest localization rates of the egfp-expressing blastomere in the ICM (6/13, 46% of ICM transgene-expressing blastocysts). This technique will have great application for multiplication of embryos of high genetic value or transgenic embryos and also with the generation of truly bovine embryonic stem cells and induced pluripotent stem cells.
Assuntos
Blastômeros/citologia , Blastômeros/metabolismo , Bovinos , Quimera/embriologia , Fase de Clivagem do Zigoto , Clonagem de Organismos/veterinária , Proteínas de Fluorescência Verde/genética , Animais , Animais Geneticamente Modificados , Bovinos/embriologia , Bovinos/genética , Bovinos/metabolismo , Fusão Celular/veterinária , Células Cultivadas , Fase de Clivagem do Zigoto/citologia , Fase de Clivagem do Zigoto/metabolismo , Fase de Clivagem do Zigoto/fisiologia , Clonagem de Organismos/métodos , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro/métodos , Proteínas de Fluorescência Verde/metabolismo , MasculinoRESUMO
Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis system using I-SceI meganuclease (intron-encoded endonuclease from S. cerevisiae) was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgene was measured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rates were higher (P < 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injection with pIS plus I-SceI after IVF increased frequency (P < 0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injection with I-SceI increased (P < 0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%, 11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.
Assuntos
Bovinos , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Fertilização in vitro/métodos , Técnicas de Transferência de Genes/veterinária , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Animais Geneticamente Modificados , Bovinos/embriologia , Bovinos/genética , Células Cultivadas , Citoplasma/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Feminino , Fertilização in vitro/veterinária , Proteínas de Fluorescência Verde/genética , Microinjeções/métodos , TransgenesRESUMO
We report an extraordinary case of collision tumor consisting of a lung adenocarcinoma and a metastatic adenoid cystic carcinoma in a 56 year-old man. He was diagnosed with a pulmonary nodule 11 years after treatment of an adenoid cystic carcinoma of the right maxillary sinus. A non-small cell carcinoma was observed when a transbronchial biopsy was performed. The other component of the nodule was only diagnosed with pathological examination of the resection specimen.
Assuntos
Adenocarcinoma/patologia , Carcinoma Adenoide Cístico/secundário , Neoplasias Pulmonares/secundário , Neoplasias do Seio Maxilar/patologia , Adenocarcinoma de Pulmão , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
The objective was to evaluate the effects of cell cycle inhibitors (6-dimethylaminopurine [DMAP], and dehydroleukodine [DhL]) on transgene expression efficiency and on mosaic expression patterns of IVF bovine zygotes cytoplasmically injected with oolema vesicles coincubated with transgene. The DNA damage induced by the transgene or cell cycle inhibitors was measured by detection of phosphorylated histone H2AX foci presence (marker of DNA double-stranded breaks). Cloning of egfp blastomeres was included to determine continuity of expression after additional rounds of cellular division. The pCX-EGFP [enhanced green fluorescent protein gene (EGFP) under the chimeric cytomegalovirus IE-chicken-ß-actin enhancer promoter control] gene plasmid (50 ng/µL) was injected alone (linear or circular exogenous DNA, leDNA and ceDNA, respectively) or associated with ooplasmic vesicles (leDNA-v or ceDNA-v). The effects of 2 mm DMAP or 1 µm DhL for 6 h (from 15 to 21 h post IVF) was evaluated for groups injected with vesicles. The DMAP increased (P < 0.05) egfp homogenous expression relative to transgene alone (21%, 18%, and 11% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively) and also increased (P < 0.05) the phosphorylated histone H2AX foci area. Expression of egfp was higher (P < 0.05) for linear than for circular pCX-EGFP, and egfp blastocyst rates were higher (P < 0.05) for groups injected with linear transgene coincubated with vesicles than for linear transgene alone (95%, 77%, 84%, and 52% for leDNA-v + DMAP, leDNA-v + DhL, leDNA-v, and leDNA, respectively). Moreover, DMAP tended to improve egfp blastocysts rates for both circular and linear transgenes. Based on fluorescent in situ hybridization (FISH) analysis, there was evidence of integration in egfp embryos. Finally, clones derived from leDNA-v + DMAP had the highest egfp expression rates (96%, 65%, and 65% for leDNA-v + DMAP, leDNA-v, and leDNA, respectively). Transgenesis by cytoplasmic injection of leDNA-v + DMAP is a promising alternative for transgenic animal production.
Assuntos
Animais Geneticamente Modificados , Bovinos/embriologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Clonagem de Organismos/métodos , Fertilização in vitro , Inibidores de Proteínas Quinases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Bovinos/genética , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos , Feminino , Fertilização in vitro/efeitos dos fármacos , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Lactonas/farmacologia , Masculino , Sesquiterpenos/farmacologia , Transgenes/genéticaRESUMO
Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development. The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.
Assuntos
Gibberella/genética , Gibberella/metabolismo , Giberelinas/biossíntese , Cruzamentos Genéticos , Genes Fúngicos , Teste de Complementação Genética , Mutagênese , Esporos Fúngicos/genética , Transformação GenéticaRESUMO
The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others.
Assuntos
Fusarium/genética , Transformação Genética , Alelos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Fúngico/genética , Conversão Gênica , Plasmídeos/genética , Recombinação GenéticaRESUMO
Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces.
Assuntos
Alquil e Aril Transferases/genética , Carotenoides/biossíntese , Genes Fúngicos , Liases Intramoleculares/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Geranil-Geranildifosfato Geranil-Geraniltransferase , Dados de Sequência Molecular , Phycomyces/enzimologia , Phycomyces/genética , Phycomyces/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Phytoene synthase, phytoene dehydrogenase and carotene cyclase are three of the four enzyme activities needed to produce the acidic carotenoid neurosporaxanthin from the precursor geranylgeranyl pyrophosphate. In the filamentous fungus Fusarium fujikuroi, these three enzyme activities are encoded by two closely linked genes, carRA and carB, oriented in the same direction in the genome. The two genes are separated by 548 bp and code for two polypeptides of 612 and 541 amino acids, respectively, which are highly similar to the homologous proteins from other filamentous fungi. The ORF of carRA contains a 96-bp insertion that is absent in the other fungal homologues. The 32 additional residues are located in one of the two repeated domains responsible for the cyclase activity in the homologous fungal proteins. We have determined the function of carRA by gene disruption. The resulting mutants were albino and had lost the ability to produce phytoene, as expected from the simultaneous loss of phytoene synthase and carotene cyclase. In the same experiments, we also found transformants in which carB had been deleted. These mutants accumulate phytoene, confirming the function of the gene previously shown by gene-targeted mutagenesis. Expression of carRA and carB is strongly induced by light. Loss of carB or disruption of the carRA ORF led to enhanced expression of the carRA gene, suggesting the existence of a feedback regulatory mechanism.