Zygotes and embryos were recovered from the reproductive tracts of CD1 and C57Bl/6 mice (Charles River Laboratories) following timed mating indicated by the presence of a copulation plug. C57Bl/6 BC5 mice (approximately 98% C57Bl/6 genotype) originally derived from chimeras bearing hybrid ES cells also were used. Zygotes were released from the fallopian tube by puncturing the ampulla, while later-stage embryos were expelled from the fallopian tube by flushing with Embryo Max M2 media (Millipore) using a syringe fitted with a short bevel 30G needle. Cumulus cells, when present, were removed by brief incubation at room temperature in Embryo Max M2 media with hyaluronidase (Millipore). Following recovery, embryos were transferred in batches to 60 mm Petri plates containing multiple 25 μL drops of Embryo Max Advanced KSOM Embryo Medium (Millipore) covered with 10 mL of mineral oil (M8410; Sigma–Aldrich). Plates were held in a humidified incubator at 37°C with 5% CO2 in air overnight prior to introducing embryos and were maintained under the same conditions.
Embryomax advanced ksom embryo medium
Manufactured by Merck Group
Sourced in United States
EmbryoMax® Advanced KSOM Embryo Medium is a laboratory product designed for the in vitro culture of embryos. It provides the necessary nutrients and growth factors to support embryo development.
Automatically generated - may contain errors
Lab products found in correlation
Trueguide sgrna, by Thermo Fisher Scientific (3 mentions)
Embryo max electroporation buffer, by Merck Group (3 mentions)
Truecut cas9 protein v2, by Thermo Fisher Scientific (3 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (2 mentions)
Cd1 and c57bl6 mice, by Charles River Laboratories (1 mentions)
M8410, by Merck Group (1 mentions)
Pregnant mare serum gonadotropin, by Merck Group (1 mentions)
Femtojet 4i microinjector, by Eppendorf (1 mentions)
H4272, by Merck Group (1 mentions)
6 protocols using embryomax advanced ksom embryo medium
1
Recovering Mouse Zygotes and Embryos
2
miR-34c Inhibitor Effects on Murine Embryos
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Female C57BL/6 mice were obtained from Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). Experimental protocols involving mice were approved by the Zhejiang University Institutional Animal Care and Research Committee (Approval # ZJU20160202), and mouse care and use were in accordance with the relevant guidelines and regulations.
The mice (6–8 weeks old) were superovulated by intraperitoneal injection with 5 IU of pregnant mare serum gonadotropin (Sigma Aldrich, St. Louis, MO, USA), followed 48 h later by injection with 5 IU of human chorionic gonadotropin (hCG, Sigma). They were then mated with C57BL/6 male mice at a ratio of 1:1. Pronucleated zygotes were harvested 20–22 h after hCG injection, microinjected with an miR-34c inhibitor or a negative-control (NC) RNA and cultured in EmbryoMax Advanced KSOM Embryo Medium (Millipore, Burlington, MA, USA) under 5% CO2 in a humidified atmosphere at 37 °C. Embryos at the two-cell, four-cell and blastocyst stages were collected 44–50, 62–64 and 88–92 h after hCG injection, respectively.
The mice (6–8 weeks old) were superovulated by intraperitoneal injection with 5 IU of pregnant mare serum gonadotropin (Sigma Aldrich, St. Louis, MO, USA), followed 48 h later by injection with 5 IU of human chorionic gonadotropin (hCG, Sigma). They were then mated with C57BL/6 male mice at a ratio of 1:1. Pronucleated zygotes were harvested 20–22 h after hCG injection, microinjected with an miR-34c inhibitor or a negative-control (NC) RNA and cultured in EmbryoMax Advanced KSOM Embryo Medium (Millipore, Burlington, MA, USA) under 5% CO2 in a humidified atmosphere at 37 °C. Embryos at the two-cell, four-cell and blastocyst stages were collected 44–50, 62–64 and 88–92 h after hCG injection, respectively.
3
CRISPR-Mediated Gene Editing in Mouse Embryos
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Fertilized oocytes at the single-cell stage were harvested from FVB/N females on E0.5 and cultured in EmbryoMax® Advanced KSOM Embryo Medium (EMD Millipore) at 37°C in an atmosphere containing 5% CO2 before and after microinjection. Microinjections were performed as previously described11 (link) and consisted of 25 ng/μl TrueGuide™ sgRNA and 50 ng/μl TrueCut™ Cas9 Protein v2 (Invitrogen) diluted in EmbryoMax® Electroporation Buffer (EMD Millipore). The sgRNA targeting T/brachyury exon 3 was the same as in our earlier publication11 (link). A maximum of 28 injected zygotes were transferred bilaterally into the infundibulum of the uterine horns of pseudopregnant CD-1 females. Surrogate females were euthanized at E10.5, and conceptuses were recovered for morphological analysis and genotyping.
4
CRISPR-Mediated Gene Editing in Mouse Embryos
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Fertilized oocytes at the single-cell stage were harvested from FVB/N females on E0.5 and cultured in EmbryoMax® Advanced KSOM Embryo Medium (EMD Millipore) at 37°C in an atmosphere containing 5% CO2 before and after microinjection. Microinjections were performed as previously described11 (link) and consisted of 25 ng/μl TrueGuide™ sgRNA and 50 ng/μl TrueCut™ Cas9 Protein v2 (Invitrogen) diluted in EmbryoMax® Electroporation Buffer (EMD Millipore). The sgRNA targeting T/brachyury exon 3 was the same as in our earlier publication11 (link). A maximum of 28 injected zygotes were transferred bilaterally into the infundibulum of the uterine horns of pseudopregnant CD-1 females. Surrogate females were euthanized at E10.5, and conceptuses were recovered for morphological analysis and genotyping.
5
CRISPR-Cas9 Zygote Microinjection
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Single-cell zygotes were harvested from plugged FVB/N donors on E0.5 and were cultured in EmbryoMax® Advanced KSOM Embryo Medium (EMD Millipore) at 37 °C in an atmosphere containing 5% CO2 before and after microinjection. Reagents to be microinjected were mixed fresh 30 min before injection and consisted of 25 ng/µl TrueGuide™ sgRNA for the gene of interest (T/brachyury) and 50 ng/µl TrueCut™ Cas9 Protein v2 (Invitrogen) diluted in EmbryoMax® Electroporation Buffer (EMD Millipore). The injection solution was stored on ice until use. Microinjections and transfer surgeries were performed as described by Behringer et al. in “Manipulating the Mouse Embryo”37 . A FemtoJet 4i Microinjector (Eppendorf) was employed for injections. Injected embryos were transplanted to the oviduct of pseudopregnant CD-1 females. A maximum of 18 injected zygotes were transferred into the infundibulum of a single uterine horn. Noon of the day of the embryo transfer transplantation surgery was designated day E0.5 of gestation. Surrogate females were allowed to carry until E9.5, when they were euthanized for embryo harvest.
6
Fetuin-B Deficiency Affects Oocyte Mechanics
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C57BL/6 fetuin-B WT (Fetub+/+, n = 25) and fetuin-B deficient (Fetub−/−, n = 14) female mice with identical genetic background were used as well as fetuin-B/ovastacin double deficient (Fetub−/−, Astl−/−, n = 54) females (FVB and C57BL/6 mixed genetic background). Oocytes were isolated as described (Dietzel et al. 2013 (link)). To examine MII oocytes individually, cumulus cells surrounding the oocytes were digested with 0.16 mg/mL hyaluronidase for 3 min (H4272, Sigma). Afterward, oocytes were washed several times in 150 µL EmbryoMax® advanced KSOM embryo medium (MR 101-D, Merck).
In the case of IVF, hyaluronic digestion of cumulus cells was omitted. Sperm of C57BL/6 fetuin-B WT (Fetub+/+, n = 5, 12–17 weeks old) mice was collected as already described in Dietzel et al. (2013) (link) and directly mixed with cumulus-oocyte complexes. Twenty-four hours after insemination, the number of two-cell embryos was evaluated and separated for E-modulus measurement and ZP2 analysis. Two-cell embryos for blastocyst development evaluation were kept in KSOM medium until 5 days post-fertilization.
In the case of IVF, hyaluronic digestion of cumulus cells was omitted. Sperm of C57BL/6 fetuin-B WT (Fetub+/+, n = 5, 12–17 weeks old) mice was collected as already described in Dietzel et al. (2013) (link) and directly mixed with cumulus-oocyte complexes. Twenty-four hours after insemination, the number of two-cell embryos was evaluated and separated for E-modulus measurement and ZP2 analysis. Two-cell embryos for blastocyst development evaluation were kept in KSOM medium until 5 days post-fertilization.
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