RESUMEN
BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.
Asunto(s)
Leche , Proinsulina , Femenino , Embarazo , Animales , Bovinos , Humanos , Animales Modificados Genéticamente/metabolismo , Proinsulina/análisis , Proinsulina/metabolismo , Leche/química , Proteínas Recombinantes/metabolismo , Insulina/análisis , Péptido Hidrolasas/metabolismoRESUMEN
PURPOSE: Despite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development. MATERIALS AND METHODS: In experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran). RESULTS: In experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 103 b-ATMSCs/mL (p = 0.051), group 104 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05). CONCLUSIONS: Co-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.
Asunto(s)
Técnicas de Cocultivo , Medios de Cultivo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Adulto , Animales , Blastocisto/efectos de los fármacos , Bovinos , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/citología , Humanos , Ratones , Proteínas de Transporte de Monosacáridos/biosíntesis , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , EmbarazoRESUMEN
The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry.
Asunto(s)
Animales Modificados Genéticamente , Vectores Genéticos/metabolismo , Glándulas Mamarias Animales/metabolismo , Proteínas de la Leche/genética , Procesamiento Proteico-Postraduccional , Transgenes , Animales , Reactores Biológicos , Bovinos , Elementos Transponibles de ADN , Femenino , Vectores Genéticos/química , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Microinyecciones , Proteínas de la Leche/metabolismo , Oocitos/citología , Oocitos/metabolismo , Embarazo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine ß-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.
Asunto(s)
Vectores Genéticos/genética , Lentivirus/genética , Leche/química , Proteínas Recombinantes/metabolismo , Animales , Bovinos , Línea Celular , Células Epiteliales , Femenino , Glándulas Mamarias Animales/citología , Leche/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , TransfecciónRESUMEN
The efficiency of in vitro fertilization (IVF) depends on the viability of spermatozoa. For capuchin monkeys (Cebus apella), in vitro capacitation of spermatozoa is challenging because of their unique seminal coagulum. Motile spermatozoa can be obtained after liquefaction of the semen coagulum in coconut water-based solution. The objective of the present study was to establish an optimal in vitro maturation (IVM) protocol for capuchin monkeys and to observe the effect of follicle stimulating hormone (FSH) and luteinising hormone (LH) on IVF and parthenogenetic activation (PA) of oocytes collected from unstimulated females. We assessed spermatozoa quality after recovery from seminal coagulum using the solution ACP-118® as an extender. Oocytes were matured in vitro for 36 or 40 h and subjected to IVF or PA by applying ionomycin combined either with 6-dimethylaminopurine (6-DMAP) or roscovitine. In total, 87% of oocytes reached metaphase II (MII) after 40 IVM and 4-cell embryo production was obtained after IVF and parthenogenesis using ionomycin/6-DMAP. ACP-118® was used successfully to harvest viable spermatozoa from semen coagulum and in the preservation of spermatozoa, which were able to fertilize oocytes in vitro.
Asunto(s)
Embrión de Mamíferos/citología , Fertilización In Vitro , Metafase/efectos de los fármacos , Oocitos/citología , Partenogénesis/fisiología , Espermatozoides/citología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Cebus , Células Cultivadas , Embrión de Mamíferos/efectos de los fármacos , Femenino , Hormona Folículo Estimulante/farmacología , Hormonas/farmacología , Hormona Luteinizante/farmacología , Masculino , Oocitos/efectos de los fármacos , Partenogénesis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Roscovitina , Espermatozoides/efectos de los fármacosRESUMEN
Ovarian cortical fragments from five adult ewes were in vitro cultured for 1, 3 or 5 days in the presence of minimum essential medium either supplemented or not by follicle-stimulating hormone (FSH) (100 ng/ml) or indole-3-acetic acid (IAA) (10, 20, 40 or 100 ng/ml), alone or in combination. After in vitro culture, ovarian fragments were submitted to follicular isolation and viability test was performed using trypan blue. Addition of IAA (10 ng/ml) to a free-FSH medium resulted in the highest percentages of viable follicles, but was progressively deleterious in higher concentrations (20, 40 and 100 ng/ml) if in absence of FSH. Follicular development was observed only when FSH was added to an IAA-free medium. In conclusion, IAA at a concentration of 10 ng/ml increases follicular survival in vitro. However, at high concentrations (20, 40 or 100 ng/ml), this auxin may be deleterious to preantral follicles, the addition of FSH to the medium being necessary.
Asunto(s)
Hormona Folículo Estimulante/farmacología , Ácidos Indolacéticos/farmacología , Folículo Ovárico/efectos de los fármacos , Animales , Supervivencia Celular , Femenino , Folículo Ovárico/citología , Ovinos , Técnicas de Cultivo de TejidosRESUMEN
OBJECTIVE: To study the protein profile of oocytes and cumulus cells from different sized follicles throughout the follicular phase and to asses the ability of oocytes to progress from the dictyate to metaphase II (MII) stage. DESIGN: Animal model study. SETTING: Five academic basic research laboratories and the National Primate Centre. ANIMAL(S): Eleven normal, cycling capuchin monkey (Cebus apella) females. INTERVENTION(S): Cumulus-oocyte complexes and denuded oocytes were recovered by antral follicle aspiration. MAIN OUTCOME MEASURE(S): Protein profile analysis of denuded or intact oocytes. RESULT(S): The protein profiles of 25 denuded or intact oocytes recovered on days 5 (six denuded, five intact), 7 (four denuded, four intact), or 9 (one denuded, five intact) of the menstrual cycle were analyzed; in a second experiment, 40 intact oocytes were cultured for 24 (n = 20) or 36 hours (n = 20). The oocytes were denuded, fixed, stained, and microscopically examined to reveal the meiotic stage. The protein profile in each compartment within the cumulus-oocyte complex varied along the follicular development with a predominance of low-molecular-weight proteins in both oocyte and cumulus cells at final stages. No differences were found in the protein profile among oocytes pertaining to different sized follicles that were in the same day of the follicular phase. Oocyte MII competence was achieved only after incubation for 36 hours, and the highest maturation rate occurred in those becoming from dominant follicles. CONCLUSION(S): Our study shows, for the first time in a New World primate species, that the proteins contained in oocytes and cumulus cells reach an identical profile in the late follicular phase. This phenomenon could be related to the oocyte's ability to progress to the MII stage.
Asunto(s)
Cebus , Células del Cúmulo/metabolismo , Fase Folicular/metabolismo , Meiosis , Oocitos/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , Femenino , Factores de TiempoRESUMEN
Os níveis de progesterona (P4) no leite sem gordura, foram determinados através do método do Radioimunoteste (RIA) em fase sólida, durante 28 ciclos estrais ajustados com duraçäo entre 20 e 24 dias (x = 22,4 ñ 1,58 dias) em 11 vacas bubalinas. Os níveis de P4 no estro variaram ente 0,33 e 0,48 ng/ml, acusando, entre o 3§ e 5§ dia do ciclo, aumento que varia de 1,3 a 1,8 ng/ml. Os níveis de hormônio aumentaram gradativamente até 2,2 e 3,2 ng/ml entre o 7§ e 9§ dia e atingiram pique máximo de 3,5 a 5,8 ng/ml entre o 11§ e 13§ dia do ciclo. A partir do 15§ e 17§ um nível de 3,1 e 4,8 ng/ml foi observado o qual decresceu para 2,3 e 2,8 ng/ml entre o 19§ e 21§ dias e voltou ao nível basal de 0,4 ng/ml no estro subseqüente