Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Autores
Kaiser, German Gustavo; Mucci, Nicolas Crescencio; Gonzalez, Vega; Sánchez, Lourdes; Parrón, José Antonio; Pérez, María Dolores; Calvo, Miguel; Aller Atucha, Juan Florencio; Hozbor, Federico Andres; Mutto, Adrián Angel
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.
Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
Fuente
Journal of dairy science 100 (3) : 1605–1617. (March 2017)
Materia
Lactoferrinas
Lisozima
Vaca
Animales Transgénicos
ELISA
Leche
Milk
Transgenic Animals
Cows
Lysozyme
Lactoferrin
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/2318

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spelling Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cowKaiser, German GustavoMucci, Nicolas CrescencioGonzalez, VegaSánchez, LourdesParrón, José AntonioPérez, María DoloresCalvo, MiguelAller Atucha, Juan FlorencioHozbor, Federico AndresMutto, Adrián AngelLactoferrinasLisozimaVacaAnimales TransgénicosELISALecheMilkTransgenic AnimalsCowsLysozymeLactoferrinLactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; EspañaFil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; ArgentinaFil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina2018-05-03T17:02:29Z2018-05-03T17:02:29Z2017-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/23180022-0302https://doi.org/10.3168/jds.2016-11173Journal of dairy science 100 (3) : 1605–1617. (March 2017)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-29T13:44:18Zoai:localhost:20.500.12123/2318instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:44:18.345INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
title Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
spellingShingle Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
Kaiser, German Gustavo
Lactoferrinas
Lisozima
Vaca
Animales Transgénicos
ELISA
Leche
Milk
Transgenic Animals
Cows
Lysozyme
Lactoferrin
title_short Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
title_full Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
title_fullStr Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
title_full_unstemmed Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
title_sort Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow
dc.creator.none.fl_str_mv Kaiser, German Gustavo
Mucci, Nicolas Crescencio
Gonzalez, Vega
Sánchez, Lourdes
Parrón, José Antonio
Pérez, María Dolores
Calvo, Miguel
Aller Atucha, Juan Florencio
Hozbor, Federico Andres
Mutto, Adrián Angel
author Kaiser, German Gustavo
author_facet Kaiser, German Gustavo
Mucci, Nicolas Crescencio
Gonzalez, Vega
Sánchez, Lourdes
Parrón, José Antonio
Pérez, María Dolores
Calvo, Miguel
Aller Atucha, Juan Florencio
Hozbor, Federico Andres
Mutto, Adrián Angel
author_role author
author2 Mucci, Nicolas Crescencio
Gonzalez, Vega
Sánchez, Lourdes
Parrón, José Antonio
Pérez, María Dolores
Calvo, Miguel
Aller Atucha, Juan Florencio
Hozbor, Federico Andres
Mutto, Adrián Angel
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Lactoferrinas
Lisozima
Vaca
Animales Transgénicos
ELISA
Leche
Milk
Transgenic Animals
Cows
Lysozyme
Lactoferrin
topic Lactoferrinas
Lisozima
Vaca
Animales Transgénicos
ELISA
Leche
Milk
Transgenic Animals
Cows
Lysozyme
Lactoferrin
dc.description.none.fl_txt_mv Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.
Fil: Kaiser, German Gustavo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Mucci, Nicolas Crescencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Gonzalez, Vega. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Sánchez, Lourdes. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Parrón, José Antonio. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Pérez, María Dolores. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Calvo, Miguel. Universidad de Zaragoza. Facultad de Veterinaria. Tecnología de los Alimentos; España
Fil: Aller Atucha, Juan Florencio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Grupo de Biotecnología de la Reproducción; Argentina
Fil: Mutto, Adrián Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina
description Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme presentin milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value. Key words: bitransgenic cow, human lactoferrin, ELISA, human lysozyme.
publishDate 2017
dc.date.none.fl_str_mv 2017-03
2018-05-03T17:02:29Z
2018-05-03T17:02:29Z
dc.type.none.fl_str_mv info:eu-repo/semantics/article
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0022-0302
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url http://hdl.handle.net/20.500.12123/2318
https://doi.org/10.3168/jds.2016-11173
identifier_str_mv 0022-0302
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
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eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv Journal of dairy science 100 (3) : 1605–1617. (March 2017)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
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instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
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