PGC line derivation and culture were carried out as described (Whyte et al., 2015 (link)). Briefly, 1 μl blood isolated from a stage 16 HH embryo (Hamburger and Hamilton, 1992 (link)) was placed in culture medium containing 1× B-27 supplement (Thermo Fisher Scientific), 0.15 mM CaCl2, 2.0 mM GlutaMax (Thermo Fisher Scientific), 1× non-essential amino acids (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol, 1× EmbryoMax nucleosides (Merck Millipore), 1.2 mM pyruvate (Thermo Fisher Scientific), 0.2% ovalbumin (Sigma) and 0.01% sodium heparin (Sigma). 25 ng/ml activin A (Peprotech), 4 ng/ml FGF2 (R&D Systems) and 5 µg/ml ovotransferrin (Sigma) were added to Avian Knockout DMEM (osmolality: 250 mOsmol/kg, 12.0 mM glucose, calcium chloride free; Thermo Fisher Scientific, a custom modification of Knockout DMEM). Chicken serum at 0.2% (Biosera) was added to this medium to produce FAOTcs medium. A male and a female PGC line were derived in FAOTcs medium and expanded to 2.5×105 cells in 5 weeks before use in targeting experiments.
Ovotransferrin
Manufactured by Merck Group
Sourced in United States
Ovotransferrin is a naturally occurring protein found in egg whites. It is a member of the transferrin family of iron-binding proteins. Ovotransferrin's core function is to bind and transport iron, which is essential for various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (6 mentions)
B27 supplement, by Thermo Fisher Scientific (5 mentions)
Ovalbumin (ova), by Merck Group (4 mentions)
Heparin sodium, by Merck Group (3 mentions)
Activin a, by Thermo Fisher Scientific (3 mentions)
Embryomax nucleosides, by Merck Group (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Pyruvate, by Thermo Fisher Scientific (2 mentions)
Avian knockout dmem, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (2 mentions)
Catalase, by Merck Group (2 mentions)
Myoglobin, by Merck Group (2 mentions)
Human serum albumin (hsa), by Merck Group (2 mentions)
Cytochrome c, by Merck Group (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Hfgf2, by R&D Systems (1 mentions)
Conalbumin, by Merck Group (1 mentions)
Lysozymec, by Merck Group (1 mentions)
Chicken serum, by Biosera (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
8 protocols using ovotransferrin
1
Derivation and Culture of Avian Primordial Germ Cells
2
Avian PGC Expansion and Gene Editing
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PGC lines were derived from individual fertile eggs, cultured in FAOT medium and expanded to 400,000 cells in 5 weeks before performing gene editing experiments38 (link). Fertile eggs bred from Hy-line layer lines were incubated for 2.5 days and then 1 μl of embryonic blood was taken from the dorsal aorta of HH stage 16 HH embryos and placed into FAOT medium. FAOT medium contains custom-made Avian Knockout DMEM (Life Technologies #041-96570 M) 1× B-27 supplement (Life Technologies #17504044), 2.0 mM GlutaMax (Life Technologies #35050-038), 1× non-essential amino acids (Life Technologies #11140050), 1× EmbryoMax nucleosides (Merck Millipore #ES-008-D), 0.1 mM β-mercaptoethanol (Life Technologies #31350010), 0.2% ovalbumin (Sigma-Aldrich A5503), 1.2 mM sodium pyruvate (Life Technologies #11360070), 0.15 mM CaCl2, 0.01% sodium heparin, 4 ng/ml h-FGF2 (R&D Systems), 50 ng/ml ovotransferrin (Sigma-Aldrich C7786) and 25 ng/ml activin A (Peprotech). PGCs were grown at 37 °C in a 5% CO2 atmosphere and fed every 48 h.
3
Chick Embryonic Blood-Derived PGC Cultivation
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PGC lines were derived and cultured in FAOT medium as described in Whyte et al.11 (link). Briefly, fertile eggs from Hyline layer lines or the Roslin Green line of transgenic chickens88 (link) were incubated for 2.5 days and then 1 μl of embryonic blood was taken from the vasculature of stage 16 HH chick embryos89 (link) and placed into FAOT medium. FAOT medium contains 1 × B-27 supplement (Thermo Fisher Scientific), 2.0 mM GlutaMax (Thermo Fisher Scientific), 1 × non-essential amino acids (Thermo Fisher Scientific), 1 × EmbryoMax nucleosides (Merck Millipore), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific), 0.2% ovalbumin (Sigma), 1.2 mM pyruvate (Thermo Fisher Scientific), 0.15 mM CaCl2, 0.01% sodium heparin (Sigma), 4 ng/ml FGF2 (R&D Systems), 25 ng/ml activin A (Peprotech) and 5 µg/ml ovotransferrin (Sigma) in Avian Knockout DMEM (osmolality: 250 mOsmol/kg, 12.0 mM glucose, calcium chloride free; Thermo Fisher Scientific, a custom modification of Knockout DMEM). PGC lines were expanded to 2.4 × 105 cells in 5 weeks before use in targeting experiments. GFP-PGCs were derived from transgenic chickens created by lentiviral methods88 (link) and maintained at the National Avian Research Facility, Midlothian, UK.
4
Protein Mix Characterization Protocol
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The seven-protein mix comprised human serum albumin (HSA), cytochrome C (bovine heart), ovotransferrin (Conalbumin, chicken egg white), myoglobin (equine heart), lysozyme C (chicken egg white), and catalase (bovine liver), all purchased individually from Sigma Aldrich (St. Louis, MO). Creatine kinase Type M (rabbit muscle) was purchased from Roche (Basel, Switzerland). The cross-linker BS3 was purchased from Thermo Scientific Pierce (Rockford, IL).
5
Protein Purification Protocols
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Human serum albumin (HSA), cytochrome C (bovine),
ovotransferrin (chicken), myoglobin (equine), and catalase (bovine)
were purchased from Sigma-Aldrich (St. Louis, MO), C3b from Complement
Technology, Inc. (Tyler, TX), creatine kinase (rabbit) from Roche
(Basel, Switzerland) and BS3 from Thermo Scientific (Rockford, IL).
ovotransferrin (chicken), myoglobin (equine), and catalase (bovine)
were purchased from Sigma-Aldrich (St. Louis, MO), C3b from Complement
Technology, Inc. (Tyler, TX), creatine kinase (rabbit) from Roche
(Basel, Switzerland) and BS3 from Thermo Scientific (Rockford, IL).
6
Derivation of PGC Fibroblast Cultures
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150,000 PGCs were incubated in 500 µl of high calcium FAOT medium containing 1.8 mM CaCl2 in fibronectin-coated or gelatin-coated wells in 24-well plates for 48 h. Subsequently, the FAOT medium was replaced with PGC fibroblast cell culture medium and then refreshed every 48 h by taking out 300 µl and adding back 300 µl of PGC fibroblast medium. Adherent fibroblast-like cells were visible with 48 h of culture in PGC fibroblast medium. Cell culture medium was refreshed every 48 h and cells were split 1:3 once they are reached 85-90% confluency in 24-well plates. PGC fibroblast-like cell culture medium contains 10% ES-grade foetal bovine serum (Life Technologies #16141061), 1% chicken serum (Biosera #CH-515), 0.1% 100x NEAA (Life Technologies #11140050), 0.1% sodium pyruvate (Life Technologies #11360070), 0.1% 100x GlutaMax (Life Technologies #35050-038), and 50 ng/ml ovotransferrin (Sigma-Aldrich C7786) in Knockout DMEM (Life Technologies #10829018). PGC fibroblast cultures were maintained at 37 °C in a 5% CO2 atmosphere.
7
Primordial Germ Cell Culture Protocol
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In this study, PGC lines from two males (W4 and W19) and one female (W2), derived from WL embryos, were used. PGC lines of two males (K8 and K10) and two females (K5 and K6), derived from
Kurokashiwa embryos, were also used. PGC lines were cultured as described by Whyte et al. (2015) [18 (link)] with slight modification. PGCs
(5 × 104 cells) were seeded in 500 μl of the culture medium in a 24-well plate (Greiner Bio-one, Stonehouse, UK). The total volume of the medium in each well was changed every 2
day. The culture medium contained avian Knockout Dulbecco’s Modified Eagle Medium (DMEM) basal medium (250 mOsm/kg, 12.0 mM glucose, and calcium-chloride-free [18 (link)]), B-27 supplement, 2.0 mM GlutaMax, 1 × non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 1 × nucleosides, 1.2 mM sodium pyruvate, 2 mg/ml ovalbumin (Sigma
Aldrich, St Louis, MO, USA), 100 μg/ml sodium heparin (Sigma Aldrich), 20 μg/ml ovotransferrin (Sigma Aldrich), 0.2% chicken serum, 25 ng/ml Human Activin A (PeproTech, Rocky Hill, NJ, USA),
and 4 ng/ml human basic fibroblast growth factor (FGF) (R&D Biosystems, Minneapolis, MN, USA). Unless otherwise specified, all reagents were purchased from Thermo Fisher Scientific
(Waltham, MA, USA).
Kurokashiwa embryos, were also used. PGC lines were cultured as described by Whyte et al. (2015) [18 (link)] with slight modification. PGCs
(5 × 104 cells) were seeded in 500 μl of the culture medium in a 24-well plate (Greiner Bio-one, Stonehouse, UK). The total volume of the medium in each well was changed every 2
day. The culture medium contained avian Knockout Dulbecco’s Modified Eagle Medium (DMEM) basal medium (250 mOsm/kg, 12.0 mM glucose, and calcium-chloride-free [18 (link)]), B-27 supplement, 2.0 mM GlutaMax, 1 × non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol, 1 × nucleosides, 1.2 mM sodium pyruvate, 2 mg/ml ovalbumin (Sigma
Aldrich, St Louis, MO, USA), 100 μg/ml sodium heparin (Sigma Aldrich), 20 μg/ml ovotransferrin (Sigma Aldrich), 0.2% chicken serum, 25 ng/ml Human Activin A (PeproTech, Rocky Hill, NJ, USA),
and 4 ng/ml human basic fibroblast growth factor (FGF) (R&D Biosystems, Minneapolis, MN, USA). Unless otherwise specified, all reagents were purchased from Thermo Fisher Scientific
(Waltham, MA, USA).
8
Synthesis of Gold Nanoparticles Using Egg White
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Chloroauric acid (HAuCl4•4H2O), sodium oleate, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, avidin, ovotransferrin, ovalbumin, ovomucoid, ovoflavoprotein, lysozyme, and oleic acid (OA), oleylamine (OAm) and 1-octadecene (ODE) were obtained from Sigma-Aldrich (St. Louis, MO, USA). FeCl3 and tert-butylamine borane (TBAB) complex solutions were purchased from Sinopharm Chemical Reagent (Shanghai, China).
Analytical-grade thiourea, naphthalene, biphenyl, cyclohexane and HPLC-grade methanol (MeOH) were all purchased from Shanghai Chemical Reagent of Chinese Medicine Group (Shanghai, China). Chicken eggs were obtained from the local market. The egg white was separated from egg yolk, and then diluted with a buffer solution (10 mM sodium phosphate buffer, pH 8.6) in 1:6 ratios and filtered through a 0.22-μm membrane prior to use. All chemicals were used without any further purification. Water used in all experiments was doubly distilled and purified by a MilliQ system (Millipore, Milford, MA, USA). The samples were injected by applying a pressure of 0.5 psi for 5 s.
Analytical-grade thiourea, naphthalene, biphenyl, cyclohexane and HPLC-grade methanol (MeOH) were all purchased from Shanghai Chemical Reagent of Chinese Medicine Group (Shanghai, China). Chicken eggs were obtained from the local market. The egg white was separated from egg yolk, and then diluted with a buffer solution (10 mM sodium phosphate buffer, pH 8.6) in 1:6 ratios and filtered through a 0.22-μm membrane prior to use. All chemicals were used without any further purification. Water used in all experiments was doubly distilled and purified by a MilliQ system (Millipore, Milford, MA, USA). The samples were injected by applying a pressure of 0.5 psi for 5 s.
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