Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P 0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year /1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year /5. No cases of maternal cannibalism were found on Year /1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year /5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P 0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year /5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.

Colony aging affects the reproductive performance of Swiss Webster females used as recipients for embryo transfer

The objective was to evaluate the influence of colony aging in a Swiss Webster (SW) outbred stock used as recipients for embryo transfer. In the first study, a retrospective analysis was performed throughout several generations during a 38-month period in 2,398 embryos transferred to 108 SW recipients. A decrease in the percentage of live pups from transferred embryos was found at the end of the period. Impairment occurred due to the incidence of maternal cannibalism that increased from 0% to 67-100% (P 0.05), while pregnancy rate (pregnant/transferred recipients) and number of pups per delivered female were not affected throughout the period (P=NS). A following study was carried out to compare the reproductive performance of SW stock vs. B6D2F1 hybrid females in a 5-year interval. The study was conducted on a total of 893 embryos transferred to 40 females (20 SW and 20 B6D2F1) in Year /1, and 514 embryos transferred to 30 females (15 SW and 15 B6D2F1) in Year /5. No cases of maternal cannibalism were found on Year /1 in any of the strains (0/10 and 0/10). However, an incidence of 44,4% (4/9) was seen on Year /5 for SW, while for B6D2F1 the incidence was 0% (0/12) (P 0.05). Further examination of the uterus showed endometrial cysts and abnormal implantation sites in SW on Year /5 but not in B6D2F1 females. In conclusion, this study reports an impairment of the reproductive performance of an early aged SW outbred stock colony mainly due to the occurrence of maternal cannibalism. This finding has important implications for embryo transfer programs conducted in mouse facilities.(AU)

From reproductive technologies to genome editing in small ruminants: an embryo’s journey

The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.

From reproductive technologies to genome editing in small ruminants: an embryos journey

The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.(AU)

Transgenesis and gene edition in small ruminants / Transgênese e edição gênica em pequenos ruminantes

This review summarizes the main achievements with the use of transgenesis and genome editingtechnologies in sheep and goats. Transgenesis, also referred to as recombinant DNA (rDNA) technology, madepossible by the first time 30 years ago the addition of novel traits from a given species into a different one. Onthe other hand, more recently genome editing appears a much more precise method of making changes to thegenome of a plant, animal, or other living organism, allowing for the addition, substitution, or deletion ofspecific nucleotides in an organisms genome. With transgenesis, the introduction of new DNA into anorganisms genome was generally without control of the site of the genome in which the insertion of that rDNAconstruct would occur. With genome editing in contrast, researchers and developers of products can makespecific changes in precise locations of the genome. This concept was absolutely improved with the novelCRISPR/Cas system, making genome edition cheaper, more efficient, easier and affordable for every Laboratoryaround the world. This revolution that originally emerged from molecular biology and passed to biomedicine,has recently been applied to livestock and agriculture. In addition, the application of this technology in sheep,goats, pigs and cattle, also has been possible by the advance of assisted reproductive technologies for embryoproduction, micromanipulation, cryopreservation and transfer. In general, multidisciplinary approaches includingbasic research and technical improvements, participation of private actors and adequate regulation should bemerged to take advantage of this potent biotechnology in different countries.(AU)

Transgenesis and gene edition in small ruminants / Transgênese e edição gênica em pequenos ruminantes

This review summarizes the main achievements with the use of transgenesis and genome editingtechnologies in sheep and goats. Transgenesis, also referred to as recombinant DNA (rDNA) technology, madepossible by the first time 30 years ago the addition of novel traits from a given species into a different one. Onthe other hand, more recently genome editing appears a much more precise method of making changes to thegenome of a plant, animal, or other living organism, allowing for the addition, substitution, or deletion ofspecific nucleotides in an organism’s genome. With transgenesis, the introduction of new DNA into anorganism’s genome was generally without control of the site of the genome in which the insertion of that rDNAconstruct would occur. With genome editing in contrast, researchers and developers of products can makespecific changes in precise locations of the genome. This concept was absolutely improved with the novelCRISPR/Cas system, making genome edition cheaper, more efficient, easier and affordable for every Laboratoryaround the world. This revolution that originally emerged from molecular biology and passed to biomedicine,has recently been applied to livestock and agriculture. In addition, the application of this technology in sheep,goats, pigs and cattle, also has been possible by the advance of assisted reproductive technologies for embryoproduction, micromanipulation, cryopreservation and transfer. In general, multidisciplinary approaches includingbasic research and technical improvements, participation of private actors and adequate regulation should bemerged to take advantage of this potent biotechnology in different countries.

Eficiencia de la transgenesis murina en el institut pasteur de Montevideo / /Murine transgenesis techniques efficiency of institute pasteur Montevideo

Los animales transgénicos son empleados en investigación biomédica para estudiar la función y regulación de los genes, como modelos de enfermedades humanas y para ensayar estrategias terapéuticas. Asimismo, la transgénesis es aplicada en animales productivos para la generación de proteínas recombinantes y la mejora animal, y para la realización de xenotrasplantes. La Unidad de Animales Transgénicos y de Experimentación del Institut Pasteur de Montevideo produce ratones transgénicos a demanda para investigadores locales y de la región. El objetivo de este trabajo es mostrar la eficiencia durante la puesta a punto de alguna de las técnicas empleadas: (i) microinyección pronuclear de ADN (Tg clásica); (ii) transgénesis mediada por lentivirus (LV); y (iii) microinyección de células madre embrionarias (ESC) en blastocistos. Los parámetros evaluados fueron: sobrevida embrionaria 30 min post-inyección, tasa de preñez, de nacimiento, de transgénesis y tasa global de transgénesis. La sobrevida embrionaria resultó en 54,6%, 78,8% y 87,8% para Tg clásica, ESC y LV, respectivamente. El número de embriones GFP (proteína verde fluorescente) + inyectados con LV resultó 42,6%. La tasa de preñez fue 54,8% y 25,0%; la tasa de nacimiento fue 21,3% y 24,0%; la tasa de transgénesis fue 4,5% y 24,1%; y la tasa de transgénesis global fue 0,2% y 0,8%, para Tg clásica y ESC, respectivamente. En suma, se produjeron 21 animales fundadores por Tg clásica, 7 animales quiméricos por inyección de ESC, y más de un 50% de embriones resultaron GFP+ con la técnica de inyección de LV. Las técnicas empleadas resultaron efectivas para obtener los modelos transgénicos solicitados. CEUA Nº 01-012.

Transgenic animals are used in biomedical research to study gene function and regulation, as human disease models, and to test therapeutic strategies. Moreover, transgenic farm animals are used for recombinant protein production, sanitary improvement, and xenotransplantation. The Transgenic and Experimental Animal Unit of Institut Pasteur Montevideo produces transgenic mice for local and regional researchers. The objective of this work is to show the efficiency of transgenesis techniques during their set up at the unit: (i) DNA pronuclear microinjection (classical Tg); (ii) lentiviral-mediated transgenesis (LV); and (iii) embryonic stem cell (ESC) microinjection in blastocysts. The parameters evaluated were: embryo survival 30 min post-injection, pregnancy rate, birth rate, transgenesis rate, and global transgenesis rate. Embryo survival was 54.6%, 78.8% and 87.8% for classical Tg, ESC and LV, respectively. The number of GFP+ embryos injected with LV was 42.6%. Pregnancy rate was 54.8% and 25.0%; birth rate was 21.3% and 24.0%; transgenesis rate was 4.5% and 24.1%; and global transgenesis rate was 0.2% and 0.8%, for classical Tg and ESC, respectively. In total, 21 founder animals were produced by classical Tg, 7 chimeric animals by ESC injection, and more than 50% GFP+ embryos were obtained using LV injection. The techniques used at the unit were effective to obtain the required transgenic models.

Eficiencia de la transgenesis murina en el institut pasteur de Montevideo / /Murine transgenesis techniques efficiency of institute pasteur Montevideo

Los animales transgénicos son empleados en investigación biomédica para estudiar la función y regulación de los genes, como modelos de enfermedades humanas y para ensayar estrategias terapéuticas. Asimismo, la transgénesis es aplicada en animales productivos para la generación de proteínas recombinantes y la mejora animal, y para la realización de xenotrasplantes. La Unidad de Animales Transgénicos y de Experimentación del Institut Pasteur de Montevideo produce ratones transgénicos a demanda para investigadores locales y de la región. El objetivo de este trabajo es mostrar la eficiencia durante la puesta a punto de alguna de las técnicas empleadas: (i) microinyección pronuclear de ADN (Tg clásica); (ii) transgénesis mediada por lentivirus (LV); y (iii) microinyección de células madre embrionarias (ESC) en blastocistos. Los parámetros evaluados fueron: sobrevida embrionaria 30 min post-inyección, tasa de preñez, de nacimiento, de transgénesis y tasa global de transgénesis. La sobrevida embrionaria resultó en 54,6%, 78,8% y 87,8% para Tg clásica, ESC y LV, respectivamente. El número de embriones GFP (proteína verde fluorescente) + inyectados con LV resultó 42,6%. La tasa de preñez fue 54,8% y 25,0%; la tasa de nacimiento fue 21,3% y 24,0%; la tasa de transgénesis fue 4,5% y 24,1%; y la tasa de transgénesis global fue 0,2% y 0,8%, para Tg clásica y ESC, respectivamente. En suma, se produjeron 21 animales fundadores por Tg clásica, 7 animales quiméricos por inyección de ESC, y más de un 50% de embriones resultaron GFP+ con la técnica de inyección de LV. Las técnicas empleadas resultaron efectivas para obtener los modelos transgénicos solicitados. CEUA Nº 01-012.(AU)

Transgenic animals are used in biomedical research to study gene function and regulation, as human disease models, and to test therapeutic strategies. Moreover, transgenic farm animals are used for recombinant protein production, sanitary improvement, and xenotransplantation. The Transgenic and Experimental Animal Unit of Institut Pasteur Montevideo produces transgenic mice for local and regional researchers. The objective of this work is to show the efficiency of transgenesis techniques during their set up at the unit: (i) DNA pronuclear microinjection (classical Tg); (ii) lentiviral-mediated transgenesis (LV); and (iii) embryonic stem cell (ESC) microinjection in blastocysts. The parameters evaluated were: embryo survival 30 min post-injection, pregnancy rate, birth rate, transgenesis rate, and global transgenesis rate. Embryo survival was 54.6%, 78.8% and 87.8% for classical Tg, ESC and LV, respectively. The number of GFP+ embryos injected with LV was 42.6%. Pregnancy rate was 54.8% and 25.0%; birth rate was 21.3% and 24.0%; transgenesis rate was 4.5% and 24.1%; and global transgenesis rate was 0.2% and 0.8%, for classical Tg and ESC, respectively. In total, 21 founder animals were produced by classical Tg, 7 chimeric animals by ESC injection, and more than 50% GFP+ embryos were obtained using LV injection. The techniques used at the unit were effective to obtain the required transgenic models.(AU)