Quimeras transitorias en la producción de embriones bovinos

La transmisión de genética superior y consecuente generación de numerosas crías idénticas de alto valor, permitiría aumentar rápidamente la cantidad y calidad del ganado vacuno. Con este fin, optimizamos la técnica de fecundación in vitro (FIV), la cual nos permitió obtener altas tasas de embriones y preñeces. La selección del toro y la realización de experimentos de FIV con semen sexado, consiguieron aumentar los rendimientos por pajuela y la producción de embriones del sexo deseado en nuestras condiciones. También exploramos un sistema eficiente de criopreservación de embriones libres de zona pelúcida (LZP) por vitrificación, con resultados de sobrevida similares al grupo control con ZP. En este sentido, mediante la desagregación de embriones en estadio de 2 y 4 células, y el posterior cultivo LZP, fue posible incrementar la cantidad inicial de embriones. Si bien la TO en presuntos cigotos de FIV permitió generar embriones de mayor tamaño, también afectó severamente tanto la competencia de los embriones como la de sus blastómeras individuales. Sin embargo, la complementación troboblástica de blastómeras más avanzadas desagregadas demostró ser una alternativa innovadora para la clonación embrionaria. Este procedimiento se realizó electrofusionando embirones producidos por FIV en estadío de 2 células, que luego se agregaron con una única blastómera transgénica, generando lo que llamamos quimeras transitorias. Al emplear los embriones aneuploides y más jóvenes fue posible dirigir el destino de cada tipo celular, orientándolos hacia los tejidos extrembrionarios (que se descartarían en el momento del nacimiento), y soportando el desarrollo de una blastómera individual destinada al macizo celular interno (MCI). Por lo tanto, durante el desarrollo de esta Tesis fue posible desarrollar un sistema completo y original de clonación embrionaria, basado en la producción, multiplicación y criopreservación de embriones bovinos LZP

Recent advances in micromanipulation and transgenesis in domestic mammals

Background: Intracytoplasmic sperm injection (ICSI) involves mechanical transfer of a single sperm cell into ooplasm. A new application has been recently found for ICSI, the production of transgenic animals. Since the birth of ‘‘Dolly’’, the first adult somatic cloned mammal, viable offspring has been produced by nuclear transfer in many species including cattle. The present review briefly summarizes our experience with ICSI and somatic cell nuclear transfer mainly to produce transgenic embryos, as well as for the generation of new micromanipulation technique. Review: We have evaluated different factors that affect SCNT and transgenesis including the chemical activator, the transfection event and the effect of recloning. Also, we included a brief description of the ICSI technique, which we used in five different species, examining its potential to produce transgenic embryos. Finally different strategies to produce transgenic animals were analyzed: ICSI- mediated gen transfer (ICSI-MGT), Injection of cumulus cell and ooplasmic vesicle incubated for 5 min with the transgene or injection of the plasmid alone. All of them were very efficient in exogenous DNA expression at embryo stages but resulted in mosaic embryos. We demonstrated that “ICSI-MGT” assisted by chemical activation is the only treatment of sperm mediated gen transfer capable to generated transgenic embryos in ovine. Besides, after ICSI-MGT, it is possible to obtain enhanced green fluorescent protein (EGFP)-expressing embryos in five diferent species: ovine, porcine, feline, bovine and equine. Our studies also established for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos, and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection of vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique. We tried a further simplification of the technique in bovine oocytes and zygotes, by intracytoplasmically injecting them with eDNA-liposomes complexes. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp–liposome was injected 16 h post-fertilization. Different approaches were assayed to reverse the mosaicism including a novel technique of gamete cloning. Our first approach consisted of the production of transgenic IVF embryos by vesicle microinjection to generate transgenic blastomeres to be used as donor cells for cloning. A high efficiency in mosaicism reversal and multiplication of transgenic embryos was attaineded. Other technique assayed was the separation of transgenic blastomeres followed by the aggregation of two-cell fused embryos or by the asynchronous younger blastomere successfully multiplied transgenic embryos, and theoretically reduces mosaicism rates in future offspring [15]. This technology can also be used to multiply embryos from animals with high genetic value. We demonstrated that a sperm and oocyte can be efficiently cloned. Green haploid androgenic blastomeres produced with the injection of a single sperm by egfp ICSI-MGT could be used to fertilized oocytes resulting in several homogeneous expressing embryos. This approach shows great potential because it allows for determination of the sex of the sperm nucleus prior to fertilization. It is also possible to clone previously transfected oocytes followed by the reconstruction of biparental bovine embryos to generate homogeneous transgene-expressing embryos. This review summarizes recent experiments in micromanipulation and gene transfer in domestic animals. The objective is not to exhaustedly describe the research done in this field but to present the promising methods recently developed or evaluated in our lab. Conclusion: Significant advancements have been made in the course of the recent years in micromanipulation and transgenesis techniques. In our lab we have been evaluating ICSI and Nuclear transfer mainly to produce transgenic embryos. We used also transgensis to apply or developed new micromanipulation technique in domestic animals linke sperm and oocyte cloning.Fil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Unidad Ejecutora de Investigaciones En Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones En Producción Animal; ArgentinaFil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Unidad Ejecutora de Investigaciones En Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones En Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Hospital Italiano; ArgentinaFil: Gambini, Andres. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Vichera, Gabriel Damian. Universidad Nacional de San Martín; ArgentinaFil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Jarazo, Javier

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis systemusing I-SceI meganuclease (intron-encoded endonuclease fromS. 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 transgenewasmeasured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rateswere 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, injectionwith 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, injectionwith 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.Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Sipowicz, P.. Universidad Nacional de San Martín. Laboratorio de Neuro y Citogenética Molecular; ArgentinaFil: Rozenblum, G. T.. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Vitullo, Alfredo Daniel. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Radrizzani Helguera, Martin. Universidad Nacional de San Martín. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentin

Recientes avances en micromanipulación y transgénesis en mamíferos domésticos

Esta revisión describe los trabajos que hemos realizado en el Laboratorio de Biotecnología Animal de la Facultad de Agronomía de la Universidad de Buenos Aires y trabajos hechos en colaboración con una empresa de biotecnología argentina. Los experimentos realizados fueron principalmente en el área de micromanipulación embrionaria y transgénesis animal. Se describen experiencias de transgénesis por transplante nuclear utilizando células genéticamente modificadas incluyendo la reclonacion de animales transgénicos. Luego se presentan resultados en que mediante la inyección intracitoplasmática de espermatozoide (ICSI) permitieron producir embriones trasgénicos en cinco especies domésticas diferentes. Otros trabajos descriptos exploran numerosas alternativas para generar transgénesis por ICSI en bovinos y ovinos. Seguidamente se analizaron varias estrategias para producir animales transgénicos entre ellas la inyección en oocitos o cigotos de células del cúmulos, vesícula ooplásmica ambas previamente incubadas durante 5 minutos con la transgen o la inyección del plásmido solo. Todos estos tratamientos fueron eficientes induciendo la expresión de ADN exógeno en embriones preimplantados. Sin embargo hubo un gran mosaiquismo en la expresión del transgen. Varias estrategias fueron analizadas para revertir el mosaiquismo incluyendo una novedosa técnica de clonación de gametos. Concluimos que múltiples métodos nuevos de micromanipulación y transgénesis están disponibles ahora para ser empleados en las especies domésticas.Academia Nacional de Agronomía y Veterinaria (ANAV

Establishment of cell-based transposon-mediated transgenesis in cattle

Transposon-mediated transgenesis is a well-established tool for genome modification in small animal models. However, translation of this active transgenic method to large animals warrants further investigations. Here, the piggyBac (PB) and sleeping beauty (SB) transposon systems were assessed for stable gene transfer into the cattle genome. Bovine fibroblasts were transfected either with a helper-independent PB system or a binary SB system. Both transposons were highly active in bovine cells increasing the efficiency of DNA integration up to 88 times over basal nonfacilitated integrations in a colony formation assay. SB transposase catalyzed multiplex transgene integrations in fibroblast cells transfected with the helper vector and two donor vectors carrying different transgenes (fluorophore and neomycin resistance). Stably transfected fibroblasts were used for SCNT and on in vitro embryo culture, morphologically normal blastocysts that expressed the fluorophore were obtained with both transposon systems. The data indicate that transpositionis a feasible approach for genetic engineering in the cattle genome.Fil: Alessio, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Fili, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Garrels, Wiebke. Institut für Nutztiergenetik; Alemania. Gottfried Wilhelm Leibniz Universität Hannover; AlemaniaFil: Forcato, Diego Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Olmos Nicotra, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Liaudat, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Savy, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Hiriart, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Talluri, Thirumala R.. Institut für Nutztiergenetik; AlemaniaFil: Owens, Jesse B.. University of Hawaii at Manoa; Estados UnidosFil: Ivics, Zoltán. Paul-Ehrlich-Institute; AlemaniaFil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Moisyadi, Stefan. University of Hawaii at Manoa; Estados UnidosFil: Kues, Wilfried A.. Institut für Nutztiergenetik; AlemaniaFil: Bosch, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Assessing Tn5 and sleeping beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

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.Instituto de BiotecnologíaFil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Gibbons, Alejandro Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Texeira, D.I.A. Universidade Estadual do Ceará. Faculdade de Veterinária; BrasilFil: Lange, F. Universidad Maimónides. Laboratorio de Clonación y Transgenesis; ArgentinaFil: Vans Landschoot, Geraldina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fil: Lange, F. Universidad Maimónides. Laboratorio de Clonación y Transgenesis; ArgentinaFil: Savy, Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Briski, Olinda. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Grueso, Esther. Paul-Ehrlich-Institute; AlemaniaFil: Ivics, Zoltán. Paul-Ehrlich-Institute; AlemaniaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina.Fil: Kues, Wilfried A. Friedrich-Loeffler-Institut; AlemaniaFil: Ferraris, S.R. Universidad Maimónides. Laboratorio de Clonación y Transgenesis; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Replication of somatic micronuclei in bovine enucleated oocytes

<p>Abstract</p> <p>Background</p> <p>Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.</p> <p>Methods</p> <p>Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. <it>In vitro</it> matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (−)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (−)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and <it>egfp</it> expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (−), Parthenogenetic (−) and <it>in vitro</it> fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05).</p> <p>Results</p> <p>All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed <it>egfp</it> expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.</p> <p>Conclusions</p> <p>We have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.</p

Production of chimeric embryos by aggregation of bovine egfp eight-cell stage blastomeres with two-cell fused and asynchronic embryos

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.Fil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Martin, R.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona-Free Nuclear Transfer

The aim of this study was to evaluate three different cloning strategies in the domestic cat (Felis silvestris) and to use the most efficient to generate wild felid embryos by interspecific cloning (iSCNT) using Bengal (a hybrid formed by the cross of Felis silvestris and Prionailurus bengalensis) and tiger (Panthera tigris) donor cells. In experiment 1, zona-free (ZP-free) cloning resulted in higher fusion and expanded blastocyst rates with respect to zona included cloning techniques that involved fusion or injection of the donor cell. In experiment 2, ZP-free iSCNT and embryo aggregation (2X) were assessed. Division velocity and blastocyst rates were increased by embryo aggregation in the three species. Despite fewer tiger embryos than Bengal and cat embryos reached the blastocyst stage, Tiger 2X group increased the percentage of blastocysts with respect to Tiger 1X group (3.2% vs 12.1%, respectively). Moreover, blastocyst cell number was almost duplicated in aggregated embryos with respect to non-aggregated ones within Bengal and tiger groups (278.3 61.9 vs 516.8 103.6 for Bengal 1X and Bengal 2X groups, respectively; 41 vs 220 60 for Tiger 1X and Tiger 2X groups, respectively). OCT4 analysis also revealed that tiger blastocysts had higher proportion of OCT4-positive cells with respect to Bengal blastocysts and cat intracytoplasmic sperm injection blastocysts. In conclusion, ZP-free cloning has improved the quality of cat embryos with respect to the other cloning techniques evaluated and was successfully applied in iSCNT complemented with embryo aggregation.Fil: Moro, L. N.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Jarazo, J. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Buemo, Carla Paola. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sestelo, A.. Jardín Botánico de Buenos Aires; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming