WO2009014410A1 - Method for producing cloned bovine embyros - Google Patents

Method for producing cloned bovine embyros Download PDF

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Publication number
WO2009014410A1
WO2009014410A1 PCT/MX2007/000085 MX2007000085W WO2009014410A1 WO 2009014410 A1 WO2009014410 A1 WO 2009014410A1 MX 2007000085 W MX2007000085 W MX 2007000085W WO 2009014410 A1 WO2009014410 A1 WO 2009014410A1
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Prior art keywords
medium
tcm
hepes
embryos
oocytes
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PCT/MX2007/000085
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Spanish (es)
French (fr)
Inventor
Juan Jesús ROA VIDAL
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Roa Vidal Juan Jesus
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Priority to PCT/MX2007/000085 priority Critical patent/WO2009014410A1/en
Publication of WO2009014410A1 publication Critical patent/WO2009014410A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/02—Breeding vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
    • C12N15/877—Techniques for producing new mammalian cloned embryos
    • C12N15/8771—Bovine embryos
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine

Definitions

  • the invention relates to methods for the cloning of embryos and more specifically with a method for producing cloned embryos of cattle.
  • somatic cells can be used immediately (Wakayama et al., 1998) or after long-term cultivation (Kubota et al., 2000)
  • the oocytes generally come from an undescribed group of females obtained from commercial sources, the traces (Prather et al., 1987, HiII et al., 2000, Kishi et al., 2000, Kubota et al ., 2000, Hermán et al., 2002, WeIIs et al., 1999, Wells et al., 1998, Edwards et al., 2001, Piedrahita et al., 2002) or a described group of live animals using ultrasound aspiration -guided) (Bruggerhoff et al., 2002).
  • the next step is to insert a somatic nucleus into the oocyte cytoplast, to restore the equivalence of an embryonic cell.
  • this is done by the fusion of the electrical induction of the somatic cell with the cytoplast (Wilmut et al., 1997, Gibbons et al., 2002, HiII et al., 2000. Kishi et al., 2000, Kubota et al., 2000, Hermán et al., 2002, Wells et al., 1999, Wells et al., 1998, Edwards et al., 2001, Piedrahita et al., 2002)
  • a somatic cell is mechanically inserted into the perivithelial space, after this the resulting coupling (the cytoplasm of the oocyte and the somatic cell) is aligned between two electrodes and with.
  • an electric current pulse for example, 2.2 kV / cm 2 for 40 ⁇ s will induce> 70% of molten couplings (Edwards et al., 1999).
  • the electrofusion depends on maintaining the contact of the somatic cell with the cytoplasm of the oocyte so that the membranes of each cytoplasm intermingle after the formation of the pore that occurs after the electric current (First and Prather, 1991).
  • Several chemical combinations have been used for activation (Gibbons et al., 2002 Susko-Parrish et al., 1994), despite the methods of choice, the activation mimics the actions of a sperm after fertilization.
  • the cloned embryos can be transferred into linked oviducts of nurse females (Wilmut et al., 1997) or cultured in the incubator for a period of time required for development to compact or blastocyst morula and transferred to the previous synchronization receptor females (Edwards et al. ., 2001).
  • Each step of the technique has a degree of efficiency whereby the sum of the% of failures gives an ultimate result in the% viability of the clone.
  • One purpose of the present invention is to propose a new method for the production of cloned bovine embryos by means of which the use of harmful agents that imbalance the osmotic medium of the mature Hoestch ovum is avoided.
  • Another objective of the invention is to provide a new method for the production of cloned embryos in which the use of the ultraviolet light transilluminator that produces mutations (DNA fractures) in the genome is avoided.
  • a further objective of the present invention is that DNA lysis is avoided by the enucleation process since the metaphysical plate does not tend to disintegrate with the use of sucrose
  • a further objective of the invention is that with the new method of cloning of bovine embryos, shortening of exposure times and high concentrations of cytokelazine B (relaxing for microtubules) is obtained since the concentration of the metaphysical plate helps to zoning the area of enucleation
  • It is another additional objective of the new method is to provide double activation with electricity and ethanol which makes a double curve of calcium ion rise for a longer period.
  • Nuclear reprogramming of somatic cells in enucleated bovine oocytes has been, since its first work presented to the scientific community, an important tool for the development of reprogrammed cells, tissues and organisms for use in the nutritional, pharmaceutical, genotransplant industry , as well as the production of transgenic animals and genetically superior organisms in the animal industry.
  • the use of mutagenic electromagnetic frequencies, as well as chemical agents for the productivity of cloned embryos has strongly impacted the viability of embryonic development, so that establishing alternatives to avoid the use of these agents has allowed us to obtain better viability and provide future development of these organisms without the intervention of alterations that may jeopardize the genomic stability of the organism.
  • the fetus is transferred to a 10 cm diameter petri dish. With sterile scissors and tweezers, the amniotic sac is opened and the fetus is changed to another petri dish containing PBS The amniotic fluid is removed with a syringe. The tail and the head are sectioned to freeze them in PBS with 10% glycerol for future molecular analyzes.
  • a - Sowing and cell culture For the culture, the MEM (sigma) medium is previously preheated with 15% fetal bovine serum and antibiotic (Amikacin 150 ⁇ g / ml and penicillin 100 ⁇ g / ml). To inactivate the trypsin, the suspension is centrifuged and the supernatant is removed. The pellet is resuspended in culture medium (MEM) and centrifuged once more at 5 min. at 700 rpm.
  • MEM culture medium
  • the pellet is resuspended in 2 ml of culture medium, this suspension is filtered through three layers of sterile gauze, the filtrate is centrifuged, resuspended in 2 ml of medium and seeded in a 10 cm petri dish and incubate at 38.5 0 C with 90% relative humidity in 5% CO 2 and filtered air.
  • the cultures are examined after 24 hours. For these cells, the number of passages is 4 per year, so their duration in culture is around day 20.
  • oocytes the 3 to 7 mm diameter follicles are aspirated using a 10 cm syringe, with a 19 G gauge needle.
  • the recovered follicular fluid is emptied into a 50 ml plastic conical tube, where it is left sediment in a water bath at 38 ° C for 15 minutes.
  • the follicular fluid is deposited in 10 cm petri dishes, where the best quality oocyte cluster (CCO) complex is collected according to its morphological classification.
  • CCO best quality oocyte cluster
  • the CCOs are placed in drops of culture medium 199 (TCM-199) supplemented with 20 mM Hepes, 0.4% bovine serum albumin, 0.2 mM sodium pyruvate and 100 U7ml penicillin, 100 ug / ml of streptomycin.
  • the CCOs are washed and subsequently transferred to the maturation medium consisting of TCM-199 medium supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 35 ug / ml FSH (Foltropin) and streptomycin penicillin at the concentration previous.
  • the boxes are transferred to the heater at a temperature of 39 0 C in an atmosphere of 5% CO 2 in air saturation greater than 90% humidity for 20 hours.
  • the oocytes are stripped with hyaluronidase in TCM-199 hepes for 5 minutes in vortex, then washed in the middle of manipulation. Oocytes with polar body, dark cytoplasm and homogeneous are selected for cloning. Before micromanipulation the oocytes are incubated in PBS medium adding 7.5 ⁇ g / ml of cytokelazine B and 0.3% of sucrose for 10 minutes. It was thawed in a cell vial at 37 ° C. DMEM was previously heated with 5% fetal bovine serum, centrifuged at 800 rpm for 15 minutes in the same medium.
  • the supernatant is discarded and resuspended 10 times in 5 ml of DMEM with 5-10% of SFB.
  • the oocytes which are passed to the plates are enucleated, in the micromanipulator the chromosomal plates are removed using a 20 micron glass pipette of internal diameter, the metaphysical plate with a minimum volume of cytoplasm is removed by ejecting a protuction in the Perivithelial space, immediately afterwards, the cytoplast is reconstructed with the fibroblast cell and the polarity of the cytoplasto-fibroblast is organized.
  • the cycloheximide is prepared for activation, 10 ⁇ l of the cycloheximide stock in 1 ml of TCM Hepes for a working concentration of 10 pg / ml. Ethanol is used at 7% in TCM Hepes medium, 5 minutes in handling medium, washed with handling medium several times, and then in TCM Hepes, 5 hours in cycloheximide (10pg / ml) in TCM Hepes, the incubators are incubated. pre-embryos in atmosphere 5% CO 2 at 39 0 C.
  • the degenerated ovules are removed and without fragmentation, the pre-embryos with segmentation are washed 3 times in TCM Hepes and passed to SOF aa, incubated for 5 days in the incubator at 38.5 0 C with a relative humidity 90% and a mixture of gases composed of 5% CO 2 , 5% O 2 and 90% Nitrogen. Embryos are evaluated on days 5 and 7 after activation.

Abstract

The invention relates to a method for producing cloned bovine embryos, avoiding the use of mutagenic electromagnetic frequencies and chemical agents, commonly used for the production of cloned embryos and strongly impacting the viability of the development of the embryo. In this way, this novel alternative enables better viability and provides a future for the development of these organisms without alterations that may endanger the genomic stability of the organism.

Description

MÉTODO PARA LA PRODUCCIÓN DE EMBRIONES CLONADOS METHOD FOR THE PRODUCTION OF CLONATED EMBRYOS
DE BOVINOOF BOVINE
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
Campo de Ia Invención.Field of the Invention.
La invención se relaciona con procedimientos para Ia clonación de embriones y mas específicamente con un método para producir embriones clonados de bovinos.The invention relates to methods for the cloning of embryos and more specifically with a method for producing cloned embryos of cattle.
Arte Previo relacionado.Related prior art.
Bovinos adultos han sido clonados usando células del cúmulos (Kato el al., 1998. Kato el al., 2000, Tani et al., 2001 ), fibroblastos (Kato et al ., 2000, Zakhartehenko et al., 1999, HiII et al 2000, Kishi et al., 2000 Kubota et al., 2000, Kasinathan et al., 2002 Heyman et al., 2001 ), granulosa (Gibbons et al., 2002, Wells et al., 1999, Wells et al., 1998, Edwards et al., 2001 Piedrahita et al., 2002), glándula mamaria (Zakhartehenko et al., 1999, Kishi et al., 2000) de músculo (Shiga et al.,1999).células del oviducto (Kato et al., 2000, Goto et al., 1999) uterinas (Kato et al., 2000) y células Sertoli (Wakayama et al. ,1998).Adult cattle have been cloned using cluster cells (Kato el al., 1998. Kato el al., 2000, Tani et al., 2001), fibroblasts (Kato et al., 2000, Zakhartehenko et al., 1999, HiII et al 2000, Kishi et al., 2000 Kubota et al., 2000, Kasinathan et al., 2002 Heyman et al., 2001), granulosa (Gibbons et al., 2002, Wells et al., 1999, Wells et al. , 1998, Edwards et al., 2001 Piedrahita et al., 2002), mammary gland (Zakhartehenko et al., 1999, Kishi et al., 2000) of muscle (Shiga et al., 1999). Oviduct cells (Kato et al., 2000, Goto et al., 1999) uterine (Kato et al., 2000) and Sertoli cells (Wakayama et al., 1998).
Después de Ia colección, las células somáticas pueden ser utilizadas inmediatamente (Wakayama et al., 1998) o después de cultivarse por largo plazo (Kubota et al., 2000) El uso del tipo de células adultas en estado de quiescencia inducida o no (Wilmut et al., 1997) o sin quiescenia (Edwards et al. ,2001 , Shiga et al., 1999, Wakayama y Yanagimachi 1999,After the collection, the somatic cells can be used immediately (Wakayama et al., 1998) or after long-term cultivation (Kubota et al., 2000) The use of the type of adult cells in a state of induced quiescence or not ( Wilmut et al., 1997) or without quiescenia (Edwards et al., 2001, Shiga et al., 1999, Wakayama and Yanagimachi 1999,
- i - Edwards et al., 2000) es requerido para producir crías clonadas de animales adultos.- i - Edwards et al., 2000) is required to produce cloned offspring of adult animals.
Para Ia obtención de embriones clonados, los ovocitos generalmente provienen de un grupo no descrito de hembras obtenidas de fuentes comerciales, los rastros (Prather et al., 1987, HiII et al., 2000, Kishi et al.,2000, Kubota et al., 2000, Hermán et al., 2002, WeIIs et al., 1999, Wells et al., 1998, Edwards et al., 2001 , Piedrahita et al., 2002) o un grupo descrito de animales vivos usando aspiración por ultrasonido-guiado) (Bruggerhoff et al., 2002). El próximo paso es insertar un núcleo somático en el citoplasto del ovocito, para restaurar Ia equivalencia de una célula embrionaria. En especies domésticas, esto se realiza por Ia fusión de la inducción eléctrica de Ia célula somática con el citoplasto (Wilmut et al., 1997, Gibbons et al., 2002, HiII et al,. 2000. Kishi et al., 2000, Kubota et al.,2000, Hermán et al., 2002, Wells et al., 1999, Wells et al., 1998, Edwards et al., 2001 , Piedrahita et al.,2002)To obtain cloned embryos, the oocytes generally come from an undescribed group of females obtained from commercial sources, the traces (Prather et al., 1987, HiII et al., 2000, Kishi et al., 2000, Kubota et al ., 2000, Hermán et al., 2002, WeIIs et al., 1999, Wells et al., 1998, Edwards et al., 2001, Piedrahita et al., 2002) or a described group of live animals using ultrasound aspiration -guided) (Bruggerhoff et al., 2002). The next step is to insert a somatic nucleus into the oocyte cytoplast, to restore the equivalence of an embryonic cell. In domestic species, this is done by the fusion of the electrical induction of the somatic cell with the cytoplast (Wilmut et al., 1997, Gibbons et al., 2002, HiII et al., 2000. Kishi et al., 2000, Kubota et al., 2000, Hermán et al., 2002, Wells et al., 1999, Wells et al., 1998, Edwards et al., 2001, Piedrahita et al., 2002)
Inicialmente, una célula somática se inserta mecánicamente en el espacio perivitelino, después de esto el acoplamiento resultante (el citoplasma del ovocito y Ia célula somática) es alineado entre dos electrodos y con. un pulso de corriente eléctrica, (por ejemplo, de 2.2 kV/cm 2 durante 40 μs inducirán > 70% de acoplamientos fundidos. (Edwards et al. ,1999).Initially, a somatic cell is mechanically inserted into the perivithelial space, after this the resulting coupling (the cytoplasm of the oocyte and the somatic cell) is aligned between two electrodes and with. an electric current pulse, (for example, 2.2 kV / cm 2 for 40 μs will induce> 70% of molten couplings (Edwards et al., 1999).
La electrofusión depende de mantener el contacto de Ia célula somática con el citoplasma del ovocito de manera tal que las membranas de cada citoplasma se entremezclan después de Ia formación del poro que se produce posterior a Ia corriente eléctrica (First y Prather, 1991). Se han usado varias combinaciones químicas para Ia activación (Gibbons et al. ,2002 Susko-Parrish et al. ,1994), a pesar de los métodos de elección, Ia activación imita las acciones de una esperma después de Ia fertilización. Pueden transferirse los embriones clonados en oviductos ligados de hembras nodrizas (Wilmut et al., 1997) o cultivarse en Ia incubadora durante en periodo de tiempo requerido para el desarrollo a mórula compacta o blastocisto y trasnferirse a las hembras receptoras previas sincronización (Edwards et al., 2001). Cada paso de Ia técnica tiene un grado de eficiencia por Io que Ia suma de los % de fallas dan un resultado ultimo en el % de viabilidad del clon.The electrofusion depends on maintaining the contact of the somatic cell with the cytoplasm of the oocyte so that the membranes of each cytoplasm intermingle after the formation of the pore that occurs after the electric current (First and Prather, 1991). Several chemical combinations have been used for activation (Gibbons et al., 2002 Susko-Parrish et al., 1994), despite the methods of choice, the activation mimics the actions of a sperm after fertilization. The cloned embryos can be transferred into linked oviducts of nurse females (Wilmut et al., 1997) or cultured in the incubator for a period of time required for development to compact or blastocyst morula and transferred to the previous synchronization receptor females (Edwards et al. ., 2001). Each step of the technique has a degree of efficiency whereby the sum of the% of failures gives an ultimate result in the% viability of the clone.
Para el desarrollo de organismos clónicos las siguientes tres técnicas son de suma importancia: A- Reprogramación de células somáticasFor the development of clonic organisms the following three techniques are of paramount importance: A- Somatic cell reprogramming
B- Maduración y enucleación de ovocito.B- Maturation and enucleation of oocyte.
C- Activación del citoplasto reconstruido.C- Activation of the reconstructed cytoplast.
SUMARIO DE LA INVENCIÓNSUMMARY OF THE INVENTION
Un propósito de Ia presente invención es proponer un nuevo método para Ia producción de embriones clonados de bovino mediante el cual se evita Ia utilización de agentes dañinos que desequilibran el medio osmótico del óvulo maduro Hoestch. Otro objetivo de Ia invención es proporcionar un nuevo método para Ia producción de embriones clonados en el que se evita Ia utilización del transiluminador de luz ultravioleta que produce mutaciones (fracturas del DNA) en el genoma. Un objetivo más de Ia presente invención es que se evita Ia lisis del DNA por el proceso de enucleación ya que el plato metafísico no tiende a disgregarse con el uso de Ia sacarosaOne purpose of the present invention is to propose a new method for the production of cloned bovine embryos by means of which the use of harmful agents that imbalance the osmotic medium of the mature Hoestch ovum is avoided. Another objective of the invention is to provide a new method for the production of cloned embryos in which the use of the ultraviolet light transilluminator that produces mutations (DNA fractures) in the genome is avoided. A further objective of the present invention is that DNA lysis is avoided by the enucleation process since the metaphysical plate does not tend to disintegrate with the use of sucrose
(agente deshidratante) en comparación al sistema tradicional. Un objetivo adicional de Ia invención es que con el nuevo método de clonación de embriones de bovino se obtiene el acortamiento de tiempos de exposición y concentraciones altas de citoquelacina B (relajante para microtúbulos) ya que Ia concentración del plato metafísico ayuda a zonificar Ia zona de enucleación.(dehydrating agent) compared to the traditional system. A further objective of the invention is that with the new method of cloning of bovine embryos, shortening of exposure times and high concentrations of cytokelazine B (relaxing for microtubules) is obtained since the concentration of the metaphysical plate helps to zoning the area of enucleation
Es otro objetivo adicional del nuevo método es proveer Ia doble activación con electricidad y etanol Io que hace una doble curva de ascenso de ion calcio por un periodo más prolongado.It is another additional objective of the new method is to provide double activation with electricity and ethanol which makes a double curve of calcium ion rise for a longer period.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
La reprogramación nuclear de células somáticas en ovocitos enucleados de bovino ha sido, desde su primer trabajo presentado a Ia comunidad científica, una herramienta importante para el desarrollo de células, tejidos y organismos reprogramados para su uso en Ia industria nutricéutica, farmacéutica, Ia del genotransplante, así como Ia producción de animales transgénicos y organismos genéticamente superiores en Ia industria animal. El uso de frecuencias electromagnéticas mutagénicas, así como agentes químicos para Ia productividad de embriones clonados ha impactado fuertemente Ia viabilidad del desarrollo embrionario por Io que el establecer alternativas para evitar el uso de estos agentes ha permitido obtener una mejor viabilidad y proveer a futuro el desarrollo de estos organismos sin Ia intervención de alteraciones que pueda poner en riesgo Ia estabilidad genómica del organismo.Nuclear reprogramming of somatic cells in enucleated bovine oocytes has been, since its first work presented to the scientific community, an important tool for the development of reprogrammed cells, tissues and organisms for use in the nutritional, pharmaceutical, genotransplant industry , as well as the production of transgenic animals and genetically superior organisms in the animal industry. The use of mutagenic electromagnetic frequencies, as well as chemical agents for the productivity of cloned embryos has strongly impacted the viability of embryonic development, so that establishing alternatives to avoid the use of these agents has allowed us to obtain better viability and provide future development of these organisms without the intervention of alterations that may jeopardize the genomic stability of the organism.
Como un paquete tecnológico que tiene un alto valor en el mercado biotecnológico por su gran aplicación y penetración en las diferentes industrias antes mencionadas, las mejoras a las diferentes secciones técnicas son relevantes puesto que cada 1% de viabilidad puede representar de varios miles o cientos de dólares como es el caso de un animal transgénico, que su precio en el mercado oscila alrededor del millón de dólares.As a technological package that has a high value in the biotechnology market due to its great application and penetration in the different industries mentioned above, the improvements to the different technical sections are relevant since each 1% of viability can represent several thousand or hundreds of dollars as is the case of a transgenic animal, whose price in the market oscillates around one million dollars.
Derivado de Io anterior, mediante Ia presente invención se propone un nuevo método para Ia producción de embriones clonados que se caracteriza por las siguientes etapas:Derived from the foregoing, by means of the present invention, a new method for the production of cloned embryos is proposed, characterized by the following stages:
1.- Colección del feto en el rastro: Se mueve el útero del animal, se limpia con alcohol, se extrae Ia placenta y se lava con PBS y antibiótico (penicilina 100 Ul/ml). El feto con su saco amniótico, se deposita en un frasco estéril con nuevo medio PBS y antibiótico a 4°C para ser transportado al laboratorio.1.- Collection of the fetus in the trail: The uterus of the animal is moved, cleaned with alcohol, the placenta is extracted and washed with PBS and antibiotic (penicillin 100 Ul / ml). The fetus with its amniotic sac is deposited in a sterile bottle with new PBS medium and antibiotic at 4 ° C to be transported to the laboratory.
2.- Preparación del feto: Se transfiere el feto a una caja petri de 10 cm de diámetro. Con tijeras estériles y unas pinzas, se abre el saco amniótico y se cambia el feto a otra caja petri que contiene PBS. El líquido amniótico se remueve con una jeringa. Se secciona Ia cola y Ia cabeza para congelarlas en PBS con 10% de glicerol para futuros análisis moleculares.2.- Preparation of the fetus: The fetus is transferred to a 10 cm diameter petri dish. With sterile scissors and tweezers, the amniotic sac is opened and the fetus is changed to another petri dish containing PBS The amniotic fluid is removed with a syringe. The tail and the head are sectioned to freeze them in PBS with 10% glycerol for future molecular analyzes.
3.- Disgregación celular: Las piezas, de tejido diseccionadas, se cortarán en pequeños trozos con ayuda de un bisturí y pinzas, los trozos se pasan a una caja petri que contiene 4 mi de solución tripsina al 0.25% y 1 mM de EDTA en PBS. Este tejido colectado y cortado, se succiona dentro y fuera de una pipeta Pasteur. Se pone a incubar 15 minutos a 38°C agitando Ia suspensión y pipeteando para facilitar Ia disgregación.3.- Cellular disintegration: The pieces, of dissected tissue, will be cut into small pieces with the help of a scalpel and tweezers, the pieces are transferred to a petri dish containing 4 ml of 0.25% trypsin solution and 1 mM of EDTA in PBS This collected and cut tissue is suctioned in and out of a Pasteur pipette. It is incubated 15 minutes at 38 ° C by stirring the suspension and pipetting to facilitate disintegration.
A - Sembrado y cultivo celular: Para el cultivo, previamente se precalienta el medio MEM (sigma) con suero fetal bovino al 15% y antibiótico (Amikacina 150 μg/ml y penicilina 100 μg/ml). Para inactivar Ia tripsina, Ia suspensión se centrifuga y se retira el sobrenadante. El pellet se resuspende en medio de cultivo (MEM) y se centrifuga una vez más a 5 min. a 700 rpm. Se resuspende el pellet en 2 mi de medio de cultivo, esta suspensión se filtra por medio de tres capas de gasa estéril, el filtrado se centrifuga, se resuspende en 2 mi de medio y se siembra en una caja de petri de 10 cm y se pone a incubar a 38.50C con 90% de humedad relativa en 5% de CO2 y aire filtrado. Los cultivos se examinan después de 24 horas. Para estas células, el número de pasajes son de 4 por Io que su duración en cultivo es alrededor del día 20.A - Sowing and cell culture: For the culture, the MEM (sigma) medium is previously preheated with 15% fetal bovine serum and antibiotic (Amikacin 150 μg / ml and penicillin 100 μg / ml). To inactivate the trypsin, the suspension is centrifuged and the supernatant is removed. The pellet is resuspended in culture medium (MEM) and centrifuged once more at 5 min. at 700 rpm. The pellet is resuspended in 2 ml of culture medium, this suspension is filtered through three layers of sterile gauze, the filtrate is centrifuged, resuspended in 2 ml of medium and seeded in a 10 cm petri dish and incubate at 38.5 0 C with 90% relative humidity in 5% CO 2 and filtered air. The cultures are examined after 24 hours. For these cells, the number of passages is 4 per year, so their duration in culture is around day 20.
A) OBTENCIÓN Y MADURACIÓN DE OVOCITOS IN VITRO DE BOVINO Los ovarios son colectados en el rastro directamente del aparato reproductivo, los ovarios se seccionaron y se transportaron al laboratorio en Ia solución salina fisiológica con penicilina a una concentración de 100,000 Ul por litro, se enjuagan y se seleccionan los ovarios para su aspiración, descartando aquellos ovarios que se encuentran en anestro.A) OBTAINING AND MATURATION OF OVOCITS IN VITRO DE BOVINO The ovaries are collected on the trail directly from the reproductive system, the ovaries are sectioned and transported to the laboratory in the physiological saline solution with penicillin at a concentration of 100,000 Ul per liter, rinsed and the ovaries are selected for aspiration, discarding those ovaries found in anestrus.
Obtención de ovocitos: los folículos de 3 a 7 mm de diámetro son aspirados utilizando una jeringa de 10 cm., con una aguja de calibre 19 G. El líquido folicular recuperado se vacía a un tubo cónico de plástico de 50 mi, donde se deja sedimentar a baño maría a 38°C durante 15 minutos. Se deposita el líquido folicular en cajas petri de 10 cm., donde son colectados el complejo cúmulos ovocito (CCO) de mejor calidad de acuerdo a su clasificación morfológica. Una vez colectados, los CCO son puestos en gotas de medio de cultivo 199 (TCM-199) suplementando con 20 mM de Hepes, 0.4% de suero albúmina bovina, 0.2 mM de piruvato de sodio y 100 U7ml de penicilina, 100 ug/ml de estreptomicina. Los CCO se lavan y posteriormente son transferidos al medio de maduración que consiste del medio TCM-199 suplementado con 10% de suero fetal bovino, 0.2 mM de piruvato de sodio, 35 ug/ml de FSH (Foltropin) y penicilina estreptomicina a Ia concentración anterior. Las cajas se transfieren a Ia estufa a una temperatura de 390C en una atmósfera de 5% de CO2 en aire y una saturación de humedad superior al 90% durante 20 horas.Obtaining oocytes: the 3 to 7 mm diameter follicles are aspirated using a 10 cm syringe, with a 19 G gauge needle. The recovered follicular fluid is emptied into a 50 ml plastic conical tube, where it is left sediment in a water bath at 38 ° C for 15 minutes. The follicular fluid is deposited in 10 cm petri dishes, where the best quality oocyte cluster (CCO) complex is collected according to its morphological classification. Once collected, the CCOs are placed in drops of culture medium 199 (TCM-199) supplemented with 20 mM Hepes, 0.4% bovine serum albumin, 0.2 mM sodium pyruvate and 100 U7ml penicillin, 100 ug / ml of streptomycin. The CCOs are washed and subsequently transferred to the maturation medium consisting of TCM-199 medium supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 35 ug / ml FSH (Foltropin) and streptomycin penicillin at the concentration previous. The boxes are transferred to the heater at a temperature of 39 0 C in an atmosphere of 5% CO 2 in air saturation greater than 90% humidity for 20 hours.
B) ENUCLEACIÓN Y RECONSTRUCCiON NUCLEARB) NUCLEAR ENUCLEATION AND RECONSTRUCTION
Inicialmente se preparan todos los medios necesarios para desnudar, manipular, fusionar, activar y cultivar. Se precalienta una alícuota de hialuronidasa a 39°C una hora antes de desnudar. Se prepara aproximadamente 25 mi de medio de manipulación a partir del stock de medio TCM más hepes adicionándole 50 μg/ml de gentamicina y 5% de suero fetal bovino (SFB) y se mantiene a 370C en Ia incubadora sin CO2. A partir de este medio de manipulación se prepara 10 mi para Ia micromanipulación adicionando 75 μl para una concentración de trabajo de 7.5 μg/ml. Después de 18 horas de maduración los ovocitos son desnudados con hialuronidasa en TCM-199 hepes durante 5 minutos en vortex, posteriormente se lavan en medio de manipulación. Se seleccionan los ovocitos con cuerpo polar, citoplasma oscuro y homogéneo para clonación. Antes de Ia micromanipulación los ovocitos son incubados en medio PBS adicionando 7.5 μg/ml de citoquelacina B y 0.3% de sucrosa durante 10 minutos. Se descongeló en vial celular a 37°C. Previamente se calentó DMEM con 5% de suero fetal bovino, se centrifuga a 800 rpm por 15 minutos en el mismo medio. Se descarta el sobrenadante y se resuspende 10 veces en 5 mi de DMEM con 5-10% de SFB. Se procede a enuclear los ovocitos los cuales se pasaron a las placas, en el micromanipulador los platos cromosómicos son removidos usando pipeta de vidrio de 20 mieras de diámetro interno, el plato metafísico con un mínimo volumen de citoplasma es removido al eyectar una protución en el espacio perivitelino, inmediatamente después, se reconstruye el citoplasto con Ia célula de fibroblasto y se procede a Ia organización de Ia polaridad del citoplasto- fibroplasto. C) ACTIVACIÓNInitially, all the necessary means to undress, manipulate, fuse, activate and cultivate are prepared. Preheats a aliquot of hyaluronidase at 39 ° C one hour before undressing. Prepare approximately 25 ml of medium from stock manipulation medium Hepes TCM more adicionándole 50 ug / ml gentamicin and 5% fetal bovine serum (FBS) and maintained at 37 0 C in CO2 incubator without Ia. From this manipulation medium, 10 ml is prepared for the micromanipulation by adding 75 μl for a working concentration of 7.5 μg / ml. After 18 hours of maturation the oocytes are stripped with hyaluronidase in TCM-199 hepes for 5 minutes in vortex, then washed in the middle of manipulation. Oocytes with polar body, dark cytoplasm and homogeneous are selected for cloning. Before micromanipulation the oocytes are incubated in PBS medium adding 7.5 μg / ml of cytokelazine B and 0.3% of sucrose for 10 minutes. It was thawed in a cell vial at 37 ° C. DMEM was previously heated with 5% fetal bovine serum, centrifuged at 800 rpm for 15 minutes in the same medium. The supernatant is discarded and resuspended 10 times in 5 ml of DMEM with 5-10% of SFB. The oocytes which are passed to the plates are enucleated, in the micromanipulator the chromosomal plates are removed using a 20 micron glass pipette of internal diameter, the metaphysical plate with a minimum volume of cytoplasm is removed by ejecting a protuction in the Perivithelial space, immediately afterwards, the cytoplast is reconstructed with the fibroblast cell and the polarity of the cytoplasto-fibroblast is organized. C) ACTIVATION
Se prepara Ia cicloheximida para Ia activación, 10 μl del stock de cicloheximida en 1 mi de TCM Hepes para una concentración de trabajo de 10 pg/ml. El etanol se utiliza al 7% en medio TCM Hepes, 5 minutos en medio de manipulación, se lava con medio de manipulación varias veces, y después en TCM Hepes, 5 horas en cicloheximida (10pg/ml) en TCM Hepes, se incuba los pre- embriones en atmósfera 5% de CO2 a 390C.The cycloheximide is prepared for activation, 10 μl of the cycloheximide stock in 1 ml of TCM Hepes for a working concentration of 10 pg / ml. Ethanol is used at 7% in TCM Hepes medium, 5 minutes in handling medium, washed with handling medium several times, and then in TCM Hepes, 5 hours in cycloheximide (10pg / ml) in TCM Hepes, the incubators are incubated. pre-embryos in atmosphere 5% CO 2 at 39 0 C.
D) DESARROLLO DE EMBRIONES CLÓNICOS IN VITROD) DEVELOPMENT OF IN VITRO CLONIC EMBRYOS
48 horas post-incubación se sacan los óvulos degenerados y sin fragmentación, los pre-embriones con segmentación se lavan 3 veces en TCM Hepes y se pasan a SOF aa, se incuba durante 5 días en Ia incubadora a 38.50C con una humedad relativa del 90% y una mezcla de gases compuesta por 5% de CO2, 5% de O2 y 90% de Nitrógeno. Los embriones se evalúan a los días 5 y 7 a partir de su activación.48 hours post-incubation the degenerated ovules are removed and without fragmentation, the pre-embryos with segmentation are washed 3 times in TCM Hepes and passed to SOF aa, incubated for 5 days in the incubator at 38.5 0 C with a relative humidity 90% and a mixture of gases composed of 5% CO 2 , 5% O 2 and 90% Nitrogen. Embryos are evaluated on days 5 and 7 after activation.
Aunque esta invención ha sido descrita en el contexto de su modalidad preferida, para los especialistas en Ia materia será evidente que el alcance de Ia presente invención se extiende más allá de Ia modalidad específicamente descrita a otras modalidades alternas y/o usos de Ia invención que sean obvias y derivables de Ia misma. Además, aunque Ia invención se ha mostrado y descrito en detalle con apego a un modo de realización ejemplif ¡cativa, algunas otras modificaciones o cambios serán claramente evidentes para especialistas en este campo, en particular tomando como base Ia descripción que antecede. Por consiguiente, se estima que pueden hacerse varias combinaciones de las características específicas y aspectos de Ia metodología aquí descrita que caerían indiscutiblemente dentro del alcance de Ia protección del método reivindicado. Habida cuenta de Io anterior, se pretende que el alcance de Ia invención no deberá estar limitado por las modalidades particularmente ejemplificadas, sino que dicho alcance deberá estar definido por una lectura razonable de las siguientes reivindicaciones. Although this invention has been described in the context of its preferred modality, it will be clear to those skilled in the art that the scope of the present invention extends beyond the modality specifically described to other alternate modalities and / or uses of the invention that are obvious and derivable from it. In addition, although the invention has been shown and described in detail in accordance with an exemplary embodiment, some other modifications or changes will be clearly apparent to specialists in this field, in particular based on the above description. Therefore, it is estimated that they can several combinations of the specific characteristics and aspects of the methodology described herein be made that would unquestionably fall within the scope of the protection of the claimed method. In view of the foregoing, it is intended that the scope of the invention should not be limited by the particularly exemplified modalities, but that said scope should be defined by a reasonable reading of the following claims.

Claims

REIVINDICACIONES
1. Un método para producir embriones clonados de bovino, que se caracteriza por las etapas de: a) obtención y maduración de ovocitos in vitro de bovino a través del siguiente procedimiento: los ovarios son colectados en el rastro directamente del aparato reproductivo, los ovarios se seccionaron y se transportaron al laboratorio en Ia solución salina fisiológica con penicilina a una concentración de 100,000 Ul por litro, se enjuagan y se seleccionan los ovarios para su aspiración, descartando aquellos ovarios que se encuentran en anestro; obtención de ovocitos: los folículos de 3 a 7 mm de diámetro son aspirados utilizando una jeringa de 10 cm., con una aguja de calibre 19 G; el líquido folicular recuperado se vacía a un tubo cónico de plástico de 50 mi, donde se deja sedimentar a baño maría a 380C durante 15 minutos; se deposita el líquido folicular en cajas petri de 10 cm., donde son colectados el complejo cúmulos ovocito (CCO) de mejor calidad de acuerdo a su clasificación morfológica; una vez colectados, los CCO son puestos en gotas de medio de cultivo 199 (TCM-199) suplementando con 20 mM de Hepes, 0.4% de suero albúmina bovina, 0.2 mM de piruvato de sodio y 100 U7ml de penicilina, 100 ug/ml de estreptomicina; los CCO se lavan y posteriormente son transferidos al medio de maduración que consiste del medio TCM-199 suplementado con 10% de suero fetal bovino, 0.2 mM de piruvato de sodio, 35 ug/ml de FSH (Foltropin) y penicilina estreptomicina a Ia concentración anterior; las cajas se transfieren a Ia estufa a una temperatura de 390C en una atmósfera de 5% de CO2 en aire y una saturación de humedad superior al 90% durante 20 horas; b) enucleación y reconstrucción nuclear mediante el siguiente procedimiento:1. A method for producing cloned bovine embryos, characterized by the stages of: a) obtaining and maturing bovine oocytes in vitro through the following procedure: the ovaries are collected directly from the reproductive system in the trail, the ovaries they were sectioned and transported to the laboratory in the physiological saline solution with penicillin at a concentration of 100,000 Ul per liter, the ovaries are rinsed and selected for aspiration, discarding those ovaries found in anestrus; obtaining oocytes: follicles 3 to 7 mm in diameter are aspirated using a 10 cm syringe, with a 19 G gauge needle; follicular fluid recovered empties a conical plastic tube 50 mi where allowed to settle on a water bath at 38 0 C for 15 minutes; Follicular fluid is deposited in 10 cm petri dishes, where the best quality oocyte cluster (CCO) complex is collected according to its morphological classification; once collected, the CCOs are placed in drops of culture medium 199 (TCM-199) supplemented with 20 mM Hepes, 0.4% bovine serum albumin, 0.2 mM sodium pyruvate and 100 U7ml penicillin, 100 ug / ml of streptomycin; The CCOs are washed and subsequently transferred to the ripening medium consisting of TCM-199 medium supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 35 ug / ml FSH (Foltropin) and streptomycin penicillin to Ia previous concentration; the boxes are transferred to the heater at a temperature of 39 0 C in an atmosphere of 5% CO 2 in air and moisture saturation greater than 90% for 20 hours; b) nuclear enucleation and reconstruction by the following procedure:
Inicialmente se preparan todos los medios necesarios para desnudar, manipular, fusionar, activar y cultivar. Se precalientauna alícuota de hialuronidasa a 390C una hora antes de desnudar; se prepara aproximadamente 25 mi de medio de manipulación a partir del stock de medio TCM más hepes adicionándole 50 μg/ml de gentamicina y 5% de suero fetal bovino (SFB) y se mantiene a 37°C en Ia incubadora sin CO2; a partir de este medio de manipulación se prepara 10 mi para Ia micromanipulación adicionando 75 μl para una concentración de trabajo de 7.5 μg/ml; después de 18 horas de maduración los ovocitos son desnudados con hialuronidasa en TCM-199 hepes durante 5 minutos en vortex, posteriormente se lavan en medio de manipulación; se seleccionan los ovocitos con cuerpo polar, citoplasma oscuro y homogéneo para clonación; antes de Ia micromanipulación los ovocitos son incubados en medio PBS adicionando 7.5 μg/ml de citoquelacina B y 0.3% de sucrosa durante 10 minutos; se descongeló en vial celular a 37°C; previamente se calentó DMEM con 5% de suero fetal bovino, se centrifuga a 800 rpm por 15 minutos en el mismo medio; se descarta el sobrenadante y se resuspende 10 veces en 5 mi de DMEM con 5-10% de SFB; se procede a enuclear los ovocitos los cuales se pasaron a las placas, en el micromanipulador los platos cromosómicos son removidos usando pipeta de vidrio de 20 mieras de diámetro interno, el plato metafísico con un mínimo volumen de citoplasma es removido al eyectar una protución en el espacio perivitelino, inmediatamente después, se reconstruye el citoplasto con Ia célula de fibroblasto y se procede a Ia organización de Ia polaridad del citoplasto-fibroplasto; c) activación conforme al siguiente proceso: se prepara Ia cicloheximida para Ia activación, 10 μl del stock de cicloheximida en 1 mi de TCM Hepes para una concentración de trabajo de 10 pg/ml; el etanol se utiliza al 7% en medio TCM Hepes, 5 minutos en medio de manipulación, se lava con medio de manipulación varias veces, y después en TCM Hepes, 5 horas en cicloheximidaInitially, all the necessary means to undress, manipulate, fuse, activate and cultivate are prepared. Aliquot of hyaluronidase precalientauna 39 0 C one hour before stripping; approximately 25 ml of handling medium is prepared from the stock of TCM medium plus hepes by adding 50 μg / ml of gentamicin and 5% of fetal bovine serum (SFB) and kept at 37 ° C in the incubator without CO 2 ; 10 ml is prepared from this manipulation medium for micromanipulation by adding 75 μl for a working concentration of 7.5 μg / ml; after 18 hours of maturation the oocytes are stripped with hyaluronidase in TCM-199 hepes for 5 minutes in vortex, then washed in the middle of manipulation; oocytes with polar body, dark cytoplasm and homogeneous are selected for cloning; before micromanipulation the oocytes are incubated in PBS medium adding 7.5 μg / ml of cytokelazine B and 0.3% of sucrose for 10 minutes; thawed in a cell vial at 37 ° C; DMEM was previously heated with 5% fetal bovine serum, centrifuged at 800 rpm for 15 minutes in the same medium; the supernatant is discarded and resuspended 10 times in 5 ml of DMEM with 5-10% of SFB; proceed to enuclear the oocytes which were passed to the plates, in the micromanipulator the chromosomal plates are removed using a 20 micron glass pipette of internal diameter, the metaphysical plate with a minimum volume of cytoplasm is removed by ejecting a protuction in the perivithelial space, immediately afterwards, the cytoplast is reconstructed with the fibroblast cell and the polarity of the cytoplasto-fibroblast is organized; c) activation according to the following process: the cycloheximide is prepared for activation, 10 μl of the cycloheximide stock in 1 ml of TCM Hepes for a working concentration of 10 pg / ml; Ethanol is used at 7% in TCM Hepes medium, 5 minutes in handling medium, washed with handling medium several times, and then in TCM Hepes, 5 hours in cycloheximide
(10pg/ml) en TCM Hepes, se incuba los pre-embriones en atmósfera 5% de CO2 a 390C; y d) desarrollo de embriones clónicos in Vitro según el siguiente proceso: 48 horas post-incubación se sacan los óvulos degenerados y sin fragmentación, los pre-embriones con segmentación se lavan 3 veces en TCM Hepes y se pasan a SOF aa, se incuba durante 5 días en Ia incubadora a 38.5°C con una humedad relativa del 90% y una mezcla de gases compuesta por 5% de CO2, 5% de O2 y 90% de Nitrógen; los embriones se evalúan a los días 5 y 7 a partir de su activación. (10pg / ml) in Hepes TCM, pre-embryos incubated in 5% CO atmosphere 2-39 0 C; and d) development of in vitro clone embryos according to the following process: 48 hours post-incubation the degenerated and fragmented ovules are removed, the pre-embryos with segmentation are washed 3 times in TCM Hepes and passed to SOF aa, incubated during 5 days in the incubator at 38.5 ° C with a relative humidity of 90% and a mixture of gases composed of 5% CO 2 , 5% O 2 and 90% Nitrogen; embryos are evaluated on days 5 and 7 after activation.
2. El método de Ia reivindicación 1 , que se caracteriza en que no se utilizan agentes dañinos que desequilibren el medio osmótico del óvulo maduro Hoestch.2. The method of claim 1, characterized in that no harmful agents are used that unbalance the osmotic medium of the mature Hoestch ovum.
3. El método de Ia reivindicación 1 , que se caracteriza en que no se utiliza transiluminador de luz ultravioleta para evitar producir mutaciones (fracturas del DNA) en el genoma.3. The method of claim 1, characterized in that ultraviolet light transilluminator is not used to avoid producing mutations (DNA fractures) in the genome.
4. El método de Ia reivindicación 1 , que se caracteriza en que se evita Ia lisis del DNA por el proceso de enucleación ya que el plato metafísico no tiende a disgregarse con el uso de Ia sacarosa (agente deshidratante) en comparación al sistema tradicional.4. The method of claim 1, characterized in that DNA lysis is avoided by the enucleation process since the metaphysical plate does not tend to disintegrate with the use of sucrose (dehydrating agent) in comparison to the traditional system.
5. El método de Ia reivindicación 1 , que se caracteriza por el acortamiento de tiempos de exposición y concentraciones altas de citoquelacina B (relajante para microtúbujos) ya que Ia concentración del plato metafísico ayuda a zonificar Ia zona de enucleación.5. The method of claim 1, characterized by the shortening of exposure times and high concentrations of cytokelazine B (relaxing for microtubules) since the concentration of the metaphysical plate helps to zonify the enucleation zone.
6. El método de Ia reivindicación 1, que se caracteriza por Ia doble activación con electricidad y etanol Io que hace una doble curva de ascenso de ion calcio por un periodo más prolongado.6. The method of claim 1, characterized by the double activation with electricity and ethanol which makes a double curve of calcium ion rise for a longer period.
7. Embriones clonados obtenidos conforme al método que se descrito en las reivindicaciones anteriores. 7. Cloned embryos obtained according to the method described in the preceding claims.
PCT/MX2007/000085 2007-07-23 2007-07-23 Method for producing cloned bovine embyros WO2009014410A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107384854A (en) * 2017-08-16 2017-11-24 安徽医科大学 The ripe optimization liquid of human oocytes endochylema

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5453366A (en) * 1990-07-26 1995-09-26 Sims; Michele M. Method of cloning bovine embryos
US6215041B1 (en) * 1997-01-10 2001-04-10 University Of Mmassachusetts Cloning using donor nuclei from a non-quiesecent somatic cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5453366A (en) * 1990-07-26 1995-09-26 Sims; Michele M. Method of cloning bovine embryos
US6215041B1 (en) * 1997-01-10 2001-04-10 University Of Mmassachusetts Cloning using donor nuclei from a non-quiesecent somatic cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BRÜGGERHOFF K. ET AL.: "Bovine Somatic Cell Nuclear Transfer Using Recipient Oocytes Recovered by Ovum Pick-Up: Effect of Maternal linege of Oocyte Donors", BIOLOGY OF REPRODUCTION, vol. 66, 2002, pages 367 - 373 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107384854A (en) * 2017-08-16 2017-11-24 安徽医科大学 The ripe optimization liquid of human oocytes endochylema
CN107384854B (en) * 2017-08-16 2020-01-07 安徽医科大学 Human oocyte cytoplasm maturation optimization liquid

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