Transgénesis: una moderna biotecnología reproductiva en animales de interés zootécnico

Los continuos avances en el conocimiento de la biología molecular han permitido un gran progreso de la ciencia, mediante la modificación genética tanto de virus, bacterias como de organismos superiores. Estos procedimientos de alta complejidad que comprenden a la ingeniería genética permiten identificar, reproducir, modificar y transferir material genético en células, tejidos u organismos. A su vez, las modernas técnicas reproductivas que han logrado incrementar progresivamente su eficiencia en los últimos años (inseminación artificial, sincronización hormonal de estros, fecundación "in vitro", fertilización asistida, transferencia de embriones, clonación, etc.), constituyen herramientas indispensables para seguir avanzando en las nuevas investigaciones referidas a la modificación genética en los animales. Se describen brevemente las diversas metodologías empleadas para la realización de transgénesis en animales de interés zootécnico.Continuous advances in the understanding of molecular biology have allowed great progress of science, through genetic modification of viruses, bacteria and higher organisms. These highly complex procedures that include genetic engineering enable to identify, reproduce, modify and transfer genetic material into cells, tissues or organisms. In turn, modern reproductive techniques that have managed to gradually increase their efficiency in recent years (artificial insemination, hormonal synchronization of estrus, fertilization "in vitro", assisted fertilization, embryo transfer, cloning, etc.) are essential tools for continue advancing in new research concerning genetic modification in animals. The various methodologies used to carry out transgenesis in animals of zootechnical interest are briefly described.Fil: Gibbons, A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Martin, R.. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cueto, M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Bruno Galarraga, María Macarena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Efficiency of Sperm-Mediated Gene Transfer in the Ovine by Laparoscopic Insemination, In Vitro Fertilization and ICSI

Abstract. Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/ plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors. Key words: Green fluorescent protein, Sheep, Transgenesis (J. Reprod. Dev. 57: [188][189][190][191][192][193][194][195][196] 2011) ransgenesis in mammals constitutes an important tool for recombinant protein production Evidence of heterologous DNA interaction with mammalian spermatozoa and subsequent delivery into the oocyte during fertilization was first reported in 1971 The ovine is a good model in the field of animal transgenesis and biotechnology due to its relatively short pregnancy period compared with the bovine. Moreover, its significant saliva and milk production make the salivary and mammary glands interesting targets for recombinant protein expression SPERM-MEDIATED GENE TRANSFER IN THE OVINE techniques using sperm incubated with DNA for a long (2 h to 17 C) or short (5 min to 0-5 C) period of time. In addition, the interaction between sperm and labeled DNA was analyzed by fluorescent microscopy in order to interpret differences between treatments. Presence of exogenous plasmid DNA in LI, IVF and ICSI embryos was determined by PCR, and localization of exogenous DNA into their nuclei was confirmed by FISH in ICSIderived embryos. Chemical activation following nontransgenic ICSI has been reported to improve bovine embryo development Materials and Methods Animal care and governmental authorization Animal usage and care treatments were approved by the Institutional Committee of Care and Use of Laboratory Animals of the Buenos Aires University (Resolution No. 2007/15). The Secretariat of Agriculture, Livestock, Fisheries and Foods of Argentina, as counseled by the Comisión Nacional de Biotecnología Argentina (CONABIA), approved the animal confinement conditions for LI (Resolution No. 250). Chemicals and media Except where otherwise indicated, all chemicals were obtained from Sigma Chemical (St. Louis, MO, USA). DNA construction The plasmid used was pCX-EGFP (kindly provided by Dr M Okabe, Osaka University, Osaka, Japan) that contains an enhanced green fluorescent protein gene (egfp) under chimeric cytomegalovirus-IE-chicken β-actin enhancer-promoter control Sperm/DNA incubation Sperm/pCX-EGFP incubation was carried out in accordance with two procedures 1) In the first procedure, long sperm/DNA incubation (Long Incubation), fresh or frozen/thawed semen (pooled from eight rams) was treated as previously described Experimental design Three independent assays were carried out in order to evaluate exogenous gene-expressing ovine embryo production following the treatments described above. For LI, we used fresh sperm subjected to long or short sperm/DNA incubation to fertilize superovulated sheep. For the IVF and ICSI experiments, frozen/thawed sperm subjected to the same sperm-plasmid incubation treatments were used to fertilize oocytes collected from slaughterhouse ovaries. In order to analyze sperm/exogenous DNA interaction, we tested labeled-DNA adhesion to sperm membranes after Long and Short Incubation with fresh and frozen/thawed sperm. We used PCR and FISH analyses to determine transgene presence and localization in these embryos. Finally, in order to establish conditions for ICSI in the ovine, this study compared three different activation treatments to assist the ICSI technique. Each experiment was replicated at least three times. In vivo embryo production Superovulation treatments: Donor adult Merino sheep (n=17) were synchronized by insertion of an intravaginal sponge (60 mg of medroxyprogesterone acetate, Syntex; Lab. Syntex, Buenos Aires, Argentina) for 14 days. During days 12-14 of treatment, donor ewes were superovulated with six decreasing doses of FSH i.m. (18 mg × 2, 14 mg × 2, 8 mg × 2, NIH-FSH-P1, Folltropin ® , Bioniche, Armidale, NSW, Australia) administered twice a day starting on the morning of Day 12, 48 h before pessary removal, and finishing 12 h after progestagen withdrawal. Progestagen removal was performed on Day 14, coinciding with the fifth FSH treatment, and a single dose of eCG (200 UI Novormon 5000, Lab. Syntex) was administered. All ewes were checked twice daily for the onset of estrus 24 h after sponge withdrawal using an adult ram. Laparoscopic insemination (LI): Ewes in estrus were fasted 12 h prior to artificial insemination and randomly assigned to LI with fresh semen from the Long Incubation (n=9), Short Incubation (n= 8) or control (n=1) treatments. Merino rams (n= 8) of proven fertil- ity were utilized as sperm donors. Ejaculates were collected using an artificial vagina, pooled in a water bath at 36 C and treated as described previously. LI was performed 12 h after estrus detection. Ewes were placed on a standard cradle for laparoscopic procedures. The surgical field, cranial to the udder, was shaved and disinfected. General anesthesia was administered with xylazine (0.5 mg/10 kg of Kensol 2%, Konig, Buenos Aires, Argentina) and ketamine (25 mg/10 kg of Ketalar; Parke-Davis, Buenos Aires, Argentina) and local anesthesia was applied (lidocaine, Frankaina 2%; FatroVonFrankel, Buenos Aires, Argentina). Thereafter, an endoscope (Richard Wolf, Knittlingen, Germany) was inserted into the abdominal cavity through a trocar approximately 10 cm cranial to the udder and 5 cm to the left side of the midline for visualization of the uterine horns. A second trocar was inserted in the right side of the abdominal wall and used to deliver 200 μl of previously incubated or not (control) fresh sperm (500 × 10 6 /ewe final concentration) into the uterine horns (100 μl each) using a cannula (Aspic for pellet insemination in sheep, 23-gauge needle, IMV; L'Aigle, France). Finally, the trocar orifices were treated with a local antibiotic solution (Young Plata, Quimagro, Buenos Aires, Argentina), and oxytetracycline (1 ml/10 kg, Hostaciclina LA; Hoechst, Dublin, Ireland) was given as preventative treatment. Embryo recovery: Six days after estrus detection, embryos were collected from donors placed under general anesthesia, as described above. In brief, embryos were surgically obtained through prepubian laparotomy. Both uterine horns were flushed using a Foley catheter. The collection medium was prewarmed (38 C) commercial Complete Flush ViGro (Bioniche, Bogart, GA, USA). All the morulae and blastocysts collected from the different groups were kept separated for examination by fluorescence microscopy, as described below. In vitro embryo production Oocyte collection and in vitro maturation: Ovaries were collected from slaughterhouses and transported to the laboratory at ambient temperature. Cumulus-oocyte-complexes (COCs) were aspirated with 21-gauge needles from follicles with a diameter of 2 to 5 mm. They were collected into Dulbecco's phosphate buffered saline (DPBS, 14287-072; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, 013/07; Internegocios, Buenos Aires, Argentina) and 2% antibiotic-antimycotic (ATB, 15240-096; Gibco). Oocytes covered with at least 3 layers of granulosa cells were selected for in vitro maturation (IVM) in bicarbonate-buffered TCM-199 (31100-035; Gibco) containing 2 mM glutamine (G-8540), 10% FBS, 10 μg/ml follicle stimulating hormone (NIH-FSH-P1, Folltropin ® , Bioniche), 0.3 mM sodium pyruvate (P2256), 100 μM cysteamine (M9768) and 2% ATB. Oocytes were incubated in 500 μl of medium under mineral oil (M8410) in 4-well dishes (Nunclon ® , Nunc, Naperville, IL, USA) in an atmosphere of 6.5% CO2 in humidified air at 39 C for 24 h. In vitro fertilization (IVF): Semen was collected from two Merino rams using an artificial vagina, and the samples were frozen by standard procedures Intracytoplasmic sperm injection (ICSI): For ICSI, oocytes were vortexed (to remove cumulus cells) for 2 min [1 mg/ml hyaluronidase (H-4272) in DPBS] and washed tree times in HEPESbuffered (H4034) TCM-199. Mature oocytes were evaluated by visualizing the first polar body and immediately used for ICSI. Frozen semen were thawed in a 37 C water bath for 30 sec and subjected to either the Long or Short Incubation described above. After incubation, less than 0.05 μl of spermatozoa was transferred to a 4-μl droplet of TALP-H with 10% v/v of polyvinylpyrrolidone (PVP, 99219; Fisher Scientific, Pittsburgh, PA, USA) a used directly for ICSI, which was performed as described previously Oocyte chemical activation Injected oocytes were immediately activated in TALP-H with 5 μM ionomycin (I24222; Invitrogen, Carlsbad, CA, USA) for 4 min and placed in TCM-199 for 3 h to allow second polar body extrusion (except for Io-EtOH activation, where the 3-h window is not necessary for polar body emission). Oocytes were subsequently treated with a) TCM-199 with 1.9 mM DMAP (D2629) for 3 h (Io-DMAP Group); b) 5 μM ionomycin followed immediately by TCM-199 with 1.9 mM DMAP for 3 h (2Io-DMAP Group); or c) TALP-H with 7% (v/v) ethanol for 5 min (Io-EtOH Group). In all the cases, inhibitors were removed by washing three times in TALP-H, and culture was continued as described below. In vitro embryo culture Presumptive IVF and ICSI zygotes (15-30 per group) were cul-191 SPERM-MEDIATED GENE TRANSFER IN THE OVINE tured in 50-μl droplets of SOF medium [31] containing 2.5% FBS and incubated at 39 C in 6.5% CO2 in air. Embryos were moved to a new droplet every 48 h. Cleavage was evaluated on day 2, and the numbers of morulae and blastocysts were evaluated on day 7 postfertilization. Evaluation of EGFP fluorescence in embryos On day 7 postfertilization, all embryos recovered in vivo and produced in vitro were briefly exposed to blue light using an excitation filter at 488 nm and an emission filter at 530 nm to determine egfp gene expression. Sperm/exogenous DNA (eDNA) interaction In order to analyze sperm/eDNA interaction, pCX-EGFP plasmid was labeled with Alexa fluor (F6257) by the Nick Translation System (18160-010; Invitrogen). Label eDNA was incubated in accordance with the long and short treatments described above. Afterwards, an aliquot of each labeled-DNA/spermatozoa complex was diluted (10 μl in 90 μl of prewarmed TALP-H medium) and centrifuged 5 min at 490 × g. Then, the supernatant was removed carefully, and the pellets were resuspended in 100 μl of TALP-H (washing procedure). Immediately, 10 μl of each sample (Fresh or Frozen-Thawed and washed or non-washed spermatozoa) were mounted between coverslips to observed under blue light the presence of label eDNA in sperm cells in order to distinguish between motile and immotile ones. Autoflorescence was discarded by checking the spermatozoa before incubation treatments and after unlabelled bovine genomic DNA fragment incubation treatments. A second experiment was performed by labeling bovine genomic DNA with rhodamine by the Nick Translation System (0.1 to 1 Kb fragments) and using it for both sperm incubation treatments. All experiments were repeated at least two times using pooled semen from two different rams. Determination of sperm viability before and after exogenous DNA incubation Fresh semen (from two rams) that had been treated according to the Long Incubation protocol were stained by incubation in TCM-199 containing 1 mg/ml of Hoechst 33258 (861405) for 2 min, both before and after pCX-EGFP addition. Then, a 10-μl aliquot was placed between coverslips to visualize spermatozoa. At least 100 spermatozoa were observed under epifluorescent microscopy (UV 380) and classified as i) damaged membrane (stained sperm cells) motile and immotile or ii) undamaged membrane (unstained sperm cells) motile and immotile. Control spermatozoa were coincubated with Hoechst, but not with plasmid. PCR analysis Morulae and blastocysts from LI, IVF and ICSI Short Incubation with and without zonae pellucidae (ZPs) were washed in PBS, transferred as 1-μl aliquots into an eppendorf tube, resuspended in 9 μl of extraction buffer (1 mg/ml proteinase K in 10 mM Tris-EDTA, V302B, Promega, Madison, WI , USA) and incubated at 56 C for 1 h. The samples were then heated at 95 C for 10 min, and 5-μl aliquots were used for PCR. The primer set sequences were 5-GAAGTTCGAGGGCGACACCTG-3 and 5-TCGTCCATGC-CGAGAGTGATC-3 for amplifying a 269 bp fragment of EGFP. The PCR reaction conditions consisted of denaturation at 95 C for 2 min, followed by 35 amplification cycles of denaturation at 94 C for 15 sec; annealing at 55 C for 15 sec and extension at 72 C for 25 sec. Cycle 35 contained an additional extension at 72 C (7 min). The positive control consisted of 3.6 -11 g/ml of pCX-EGFP plasmid, and the negative control was distilled water. The ZP was removed by 5 min incubation in 1.5 mg/ml pronase (P8811) in TALP-H, followed by careful washing five times in 3 ml of TALP-H. Determination of the cell number in blastocysts or spermatozoa bound to embryos Embryos were stained (2 min) in TCM-199 containing 1 mg/ml of Hoechst 33342 (B2261) and immediately mounted between coverslips to visualize spermatozoa bound to the ZP (LI embryos) or to count total nuclei (ICSI blastocysts) using epifluorescent microscopy (UV 380). Fluorescence in situ hybridization (FISH) Ovine 8-cell embryos were incubated for 20 h with 0.1 μg/ml demecolcine (D1925). Afterwards, the embryos were treated in a hypotonic solution (1% Na citrate in distilled water for 10 min) and fixed on a poly-L-lysine-coated slide with 3:1 methanol-acetic acid solution. Indirect labeled signals of FITC anti-mouse (F6257) and anti-digoxigenin (D8156), which bind the digoxigenin-labeled pCX-EGFP (5.5 kb) probe, were labeled using the Nick Translation System (18160-010; Invitrogen). The total DNA was counterstained with DAPI. Images of each cell and their signals were recorded with an Optronics camera (CCD; Optronics, Goleta, CA, USA). Data analysis Embryo development and fluorescent expression were compared by Fisher's exact test analysis. Differences in total cell number or stained sperm were analyzed using a one-way ANOVA test. For statistical analyses, the SAS program was used Results Laparoscopic insemination, in vitro fertilization and intracytoplasmic sperm injection A pool of semen collected from eight rams and incubated (long or short treatment) with pCX-EGFP plasmid was used to inseminate seventeen ewes. After six days, LI embryos were recovered through laparotomy and uterine flushing. Fertilization rates were not statistically different betweens Long and Short Incubation treatments and the control group (90.4, 98.5 and 100% respectively). None of the 78 collected embryos (38 and 40 for the long and short procedures, respectively) were fluorescent after observation under blue light One hundred and sixty-two oocytes were fertilized by IVF with spermatozoa previously incubated with pCX-EGFP plasmid in the long or short treatments. Plasmid incubation groups were not different from the controls in terms of cleavage (long, short and control: 86.8, 87.3 and 88.4%, respectively) or embryonic development A total of 107 oocytes were injected with spermatozoa incubated with pCX-EGFP plasmid. Independent of incubation treatment, high embryo development and transgene expression rates were obtained Sperm/exogenous DNA interaction Plasmid DNA attachment to the plasma membrane was observed in most of the frozen-thawed spermatozoa subjected to the Long and Short ( Determination of sperm viability before and after exogenous DNA incubation The percentage of Fresh spermatozoa with a damaged membrane did not differ before (33/110; 33.0%) or after pCX-EGFP addition (43/110; 39.0%) for the Long Incubation treatment. However, both results differed significantly with respect to the control group (26/120, 21.6%; P<0.05). All stained sperm cells (membrane damaged) were immotile. PCR analyses PCR analysis showed and egfp amplification product for LI whole embryos (with a ZP). Nevertheless, when LI embryos were released from their ZPs by pronase treatment, no egfp PCR product was visible in any of the embryos analyzed. For IVF, no embryos (with or without a ZP) showed PCR amplification products. On the other hand, all ICSI embryos showed amplification of the transgene In vitro development of transgenic-ICSI ovine embryos assisted by different chemical activation treatments In total, 304 ovine oocytes were injected with spermatozoa incubated with pCX-EGFP plasmid. All ICSI activation protocols were capable of producing blastocysts FISH analyses FISH analysis displayed various possible integration loci in eight of twelve (66.6%) ovine 8-cell stage embryos Discussion After both the long and short sperm/exogenous DNA (eDNA) incubation treatments, LI embryos did not express egfp transgene Several aspects of spermatozoa/DNA interaction were studied. Both fresh and frozen/thawed spermatozoa bound eDNA, as has been found in mice and some domestic species [35-37; for a review, see 38]. Labeled DNA signals were detected on the surfaces of all fresh and frozen-thawed sperm after both treatments. However, in agreement with observations in the bovine We saw that the fresh spermatozoa that maintained exogenous attached DNA were nonmotile. Unexpectedly, embryos produced by LI (with fresh sperm) showed amplification of the transgene by PCR analysis. Nevertheless, when the PCR analysis was performed on ZP-free embryos, we did not find a specific transgene band. In order to clarify these results, we stained LI embryos with Hoechst. We observed numerous spermatozoa adhered to the ZP in 100% of the stained recovered embryos, verifying that PCR amplification was derived from spermatozoa attached to the ZP and not from the embryos. This raised the question of how spermatozoa carrying the transgene arrived at the ZP. We propose two hypotheses. 1) The first is that a small proportion of motile spermatozoa carrying the transgene were able to reach the oocytes, but none of them were able to fertilize it, and 2) the second is that nonmotile spermatozoa carrying the transgene remained in the uterine horns and could adhere to the ZP during embryo migration to the uterus. The first hypothesis has been reported in pigs, where a low percentage of motile spermatozoa (1-3%) carried eDNA, although non transgene-expressing-embryos were detected SPERM-MEDIATED GENE TRANSFER IN THE OVINE the results were independent of ZP presence. The three different activation treatments evaluated induced blastocyst development after ICSI. In order to replace the activating effect produced by piezoelectric microinjection Simple PCR amplification or RT-PCR can be used to detect the presence of a transgene, but they cannot determine its localization. An alternative technique, fluorescent in situ hybridization (FISH), can be used for this purpose. For ICSI embryos, FISH analysis at interphase found possible signals inside the nucleus compatible with at least two or four integration loci In conclusion, under the conditions studied, ICSI was the only method of fertilization to successfully produce exogenous geneexpressing ovine embryos, and the Short Incubation treatment enhanced transgene expression percentages. In addition, we found it necessary to carefully remove the ZP from embryos in order to avoid false positives by PCR. To our knowledge, this is the first study to explain possible failures involved in SMGT by LI or IVF in the ovine. The results obtained in this work demonstrated that sperm-mediated gene transfer by ICSI could be useful for inducing exogenous gene expression in ovine embryos

Activación de genes endógenos humanos de precursores óticos y de células ciliadas del oído interno mediante CRISPR-VP160

ntroducción: Últimas investigaciones en la biología celular y molecular del oído interno han aportado evidencias que apoyan nuevas modalidades terapéuticas en medicina regenerativa para restablecer la audición. Recientemente se ha utilizado elsistemaCRISPR-VP160paramejorarlosensayos de reprogramación celular de manera in vitro e in vivo. CRISPR-VP160 se compone de una endonucleasadesactivada(dCas9)fusionadacondominios de activacióndela transcripción (VP160),queutilizan un ARN guía para localizar un gen target y activarlo. El objetivo de este trabajo fue evaluar si el sistemaCRISPR-VP160puede activarlaexpresión de genes del oído interno (SOX2, ATOH1, POU4F3 Y GFI1)enlalíneacelularHEK293T. Material y método: Se diseñaron cuatro ARN guías que reconocieran al promotor de cada gen target, usando la herramienta de diseño CRISPR (Feng ZhangLab,MIT).LosARNguíasseclonaronenel plásmidoAddgene#47108yselipofectaronjuntoal plásmido dCas9-VP160 (Addgene#48226), en una relación de 1:1 en las células HEK293T. Las células del grupo control fueron cotransfectadas con el plásmidodCas9-VP160 yunplásmido guía vacío. Los resultados fueron analizados por RT-qPCR al día 4 post-lipofección. Resultados:ElsistemaCRISPR-VP160activósignificativamente a SOX2, ATOH1 y POU4F3(p<0.05), mientras que no se observó una activación significativa de GFI1, fenómeno que podría explicarse por la expresión preexistente de este gen en la línea celular. Conclusiones: En un futuro, el sistema CRISPRVP160 podría emplearse para mejorar las estrategias de reprogramación de células, para desarrollar nuevas terapias celulares en pacientes hipoacúsicos.Introduction: Recent researches in cell and molecular biology of the inner ear bring evidence that helps the development of new therapeutic practice in regenerative medicine to restore hearing. The CRISPR system has been used to improve cell reprogramming assays both in vitro and in vivo. This consists of the inactive DNA-nuclease Cas9 (dCas9)fusedtoactivationdomainsandco-expressedsingleguideRNAs(sgRNAs)thataredesigned to hybridize a target sequence. Combined, these elements recognize a target locus and activate a specific gene. The objective of this work was to evaluate whether CRISPR-on system fused with transcriptional activators (dCas9-VP160) could activate geneexpressionofinnereargenes(SOX2,ATOH1, POU4F3andGFI1)inHEK293Tcellline. Material and method: Four guides were designed to recognize the proximal promoter of each target gene, using the CRISPR design tool (Feng Zhang Lab,MIT).ThesgRNAswereclonedintoplasmid Addgene#47108andlipofectedtogetherwithplasmiddCas9-VP160(Addgen#48226),atamassratio of 1:1in HEK293T cells. Control group was co-lipofectedwithplasmiddCas9-VP160andanempty guide plasmid. The results were analyzed by RTqPCR at day 4 post-lipofection. Results: The CRISPR-VP160 system significantly activated SOX2, ATOH1 and POU4F3 (p<0.05), whereas it did not detect a significant activation of GFI1. This phenomenon could be explained by the preexisting expression of this gene in HEK293T cells. Conclusions:Inthefuture,theCRISPR-VP160system could be used to improve reprogramming strategies of cells, to develop new cellular therapies in hearing-impaired patients.Fil: Perez Raffo, Gabriela. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Vigezzi, Lucía. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Giménez, Carla Alejandra. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Boccio, Carlos Mario. Hospital Italiano; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentin

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

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

Evaluation of a lyophilized CRISPR-Cas12 assay for a sensitive, specific, and rapid detection of SARS-CoV-2

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.Instituto de VirologíaFil: Curti, Lucía Ana. CASPR Biotech; Estados UnidosFil: Primost, Ivana. Hospital Municipal de Trauma y Emergencias Dr. Federico Abete. Genetics and Molecular Biology Laboratory; ArgentinaFil: Valla, Sofia. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Investigaciones y Transferencia del Noroeste de la Provincia de Buenos Aires (CITNOBA). Centro de Investigaciones Básicas y Aplicadas (CIBA); ArgentinaFil: Valla, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ibañez Alegre, Daiana. Universidad Nacional de Misiones. Instituto de Biología Subtropical. Laboratorio Grupo de Investigación en Genética Aplicada (GIGA); ArgentinaFil: Ibañez Alegre, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olguin Perglione, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Olguin Perglione, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Repizo, Guillermo Daniel. CASPR Biotech; Estados UnidosFil: Lara, Julia. CASPR Biotech; Estados UnidosFil: Parcerisa, Ivana. CASPR Biotech; Estados UnidosFil: Palacios, Antonela. CASPR Biotech; Estados UnidosFil: Llases, María Eugenia. CASPR Biotech; Estados UnidosFil: Rinflerch, Adriana. Universidad Nacional de Misiones. Instituto de Biología Subtropical. Laboratorio Grupo de Investigación en Genética Aplicada (GIGA); ArgentinaFil: Rinflerch, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barrios, Melanie. Universidad de Buenos Aires. Instituto de Producción Agropecuaria; ArgentinaFil: Pereyra Bonnet, Federico. CASPR Biotech; Estados UnidosFil: Gimenez, Carla Alejandra. CASPR Biotech; Estados UnidosFil: Marcone, Débora Natalia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología, Biotecnología y Genética. Cátedra de Virología; ArgentinaFil: Marcone, Débora Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A simple vitrification technique for sheep and goat embryo cryopreservation

The aim of this study was to evaluate pregnancy and embryo survival rate of vitrified in vivo produced Merino sheep and Criolla goat (morulae and blastocysts) embryos, using the plastic tips of micropipettes, as containers (Cryo-tips). The embryos were exposed, at room temperature, to two successive equilibration solutions for a period of 5 min and then to a vitrification solution (VS) for 30 s. Then embryos were then loaded in 1 μl VS, into a plastic micropipette tip, and plunged into liquid nitrogen. On thawing, the embryos were warmed (37 °C) and placed into cryoprotectant dilutions (three-step-process). In the ovine, the morula and blastocyst pregnancy rates (47.1% vs 50%) and embryo survival rates (41.2% vs 50%) recorded were similar for both embryonic stages. Unlike the sheep, no pregnancies were recorded in goat vitrified/thawed morulae embryos, following transfer. However, in contrast, goats receiving blastocysts recorded high rates of pregnancy and embryo survival (64% and 64%, respectively). This technique allows for easy handling of cryopreserved embryos, is simple and efficient in both ovine embryo stages and also for goat vitrified blastocysts. The technique has definite potential application.Fil: Gibbons, A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Cueto, M. I.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

IPSCs: A Minireview from bench to bed, including organoids and the crispr system

When Dolly the sheep was born, the first probe into an adult mammalian genome traveling back in time and generating a whole new animal appeared. Ten years later, the reprogramming process became a defined method of producing induced pluripotent stem cells (iPSCs) through the overexpression of four transcription factors. iPSCs are capable of originating virtually all types of cells and tissues, including a whole new animal. The reprogramming strategies based on patient-derived cells should make the development of clinical applications of cell based therapy much more straightforward. Here, we analyze the current state, opportunities, and challenges of iPSCs from bench to bed, including organoids and the CRISPR system.Fil: Orqueda, Andres Javier. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Italiano. Instituto Universitario - Escuela de Medicina; ArgentinaFil: Giménez, Carla Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Italiano. Instituto Universitario - Escuela de Medicina; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Italiano. Instituto Universitario - Escuela de Medicina; Argentin

High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation

In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.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; ArgentinaFil: Pereyra Bonnet, Federico Alberto. 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, Rafael. 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

196 Fertilization Rate in Superovulated Criolla Goats Following Artificial Insemination or Natural Mating

The Criolla breed is a local genetic resource, distributed in Patagonia, Argentina, whose primary production is meat. In the Criolla goat, efforts were made to locate productively superior males and to conserve their genetic material. Studies were carried on the feasibility of obtaining superior offspring through the implementation of embryo transfer programs. We assessed the fertilization rate and embryo production following AI with frozen semen or natural mating in Criolla goats subjected to a superovulation program. During the breeding season (May, 41° S), 26 Criolla goats were treated for oestrus with sponges (60 mg of medroxyprogesterone acetate, Progespon®, Syntex, Buenos Aires, Argentina) placed for 13 days. Goat donors were superovulated with a total of 80 mg of porcine (p)FSH (Folltropin V®, Bioniche, Ontario, Canada) every 12 h in 6 decreasing doses (18, 18, 14, 14, 8, and 8 mg) during the last 3 days of progestagen treatment. A dose of 125 μg of cloprostenol (Estrumate®, Schering-Plough, Quebec, Canada) was given in conjunction with the first dose of pFSH. Oestrus detection was performed every 12 h, starting at 24 h after sponge removal. Females were considered to be in oestrus if they passively accepted buck mounting. Goats were randomly assigned to the following treatments: (1) natural mating (NM, n = 12): donors detected in oestrus were individually mated with one proven fertile buck at oestrus and 12 h post-oestrus; does were remove from the male in between (Conventionally accepted treatment); (2) AI (n = 14): donors detected in oestrus were inseminated 12 ± 2 h after the onset of oestrus by laparoscopy with frozen-thawed semen (200 × 106 spermatozoa) from the same fertile buck. Embryo recovery was done by surgical prepubic laparotomy at Day 8 after sponge removal. Superovulation response was estimated by counting the number of corpora lutea (CL). Analysis of variance was used to compare fertilization rate (total number of embryos recovered for each animal, expressed as a proportion of the total number of embryos/oocytes recovered) and embryo production between treatments. Results were expressed as mean ± SEM. Statistical significance was accepted at P  0.05). In conclusion, fertilization rates did not differ following laparoscopic insemination with frozen semen compared to natural mating in superovulated Criolla goats.Fil: Cueto, M. I.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte; ArgentinaFil: Fernández, J.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte; ArgentinaFil: Bruno Galarraga, María Macarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte; ArgentinaFil: Gibbons, A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte; Argentin