生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Summary

这个视频协议演示了用于产生转基因的方法<em>非洲爪蟾</em>由引进到精子细胞核转基因细胞核移植到未受精卵。

Abstract

稳定整合到爪蟾基因组克隆的基因产物，是必要的控制表达的时间和地点，在胚胎发育的后期阶段基因表达，并定义如何增强子和启动子调节胚胎中的基因表达。这里展示的协议，可以用来有效地生产转基因爪蟾胚胎。这种转基因的方法包括三个部分：1。精子细胞核分离成年X.蟾睾丸治疗溶血卵磷脂，permeabilizes精子质膜。 2。卵提取物准备，由低速离心此外，钙，造成提取的进展细胞周期相间，和高速离心分离相间细胞质。 3。核移植：细胞核和提取物相结合，与线性质粒DNA限制性内切酶的转基因和少量引进。在很短的反应，鸡蛋中提取部分decondenses精子染色质和限制性内切酶产生的染色体断裂，促进到基因组的基因重组。经处理后的精子核，然后移植到未受精卵。转基因的整合，通常会出现之前的第一等，由此产生的胚胎嵌合胚胎卵裂。没有任何需要繁殖下一代，允许转基因胚胎的新一代高效，快速的启动子和基因功能分析，可以分析这些胚胎。成人X。蟾此过程还通过生殖细胞传播的转基因和可用于产生转基因动物用于多种用途的线。

Protocol

这种转基因方法修改后的版本，最初在1和2。 A.精子细胞核的准备此核的制备方法是改编自美利3，但蛋白酶抑制剂已被省略，因为它们与干扰卵子与精子细胞核移植的后续发展。分装冷冻于-80 ° C，可使用约6个月移植。 所有的解决方案应准备，并置于冰上开始前的准备。 麻醉1-2成年男性X 。 pit…

Discussion

对于每一个转基因构造进行测试，我们一般移植到500-1000鸡蛋核;在这个规模，我们可以产生转基因胚胎的表达每天10个不同的结构，取决于有多少女性是诱导产卵。在这些移植中，约三分之一的鸡蛋切割和60-80％，这些切割胚胎正常继续通过原肠。根据使用条件的反应，这些胚胎的10-50％之间，表达转基因利益。因此，一旦这个过程是在实验室建立的，它有可能为一名工人在一天的不同质粒表达的?…

Materials

A.Sperm nuclei preparation

Reagents:

1. 1X MMR (2mM CaCl₂, 5mM HEPES, pH7.5, 2mM KCl, 1mM MgCl₂, 100mM NaCl).
2. 0.1% Tricaine Methanesulfonate (MS222, aminobenzoic acid ethyl ester, Sigma A-5040), 0.1% sodium bicarbonate. Dissolve in water.
3. 2X Nuclear Preparation Butter (NPB). On the day of the sperm nuclei preparation, make up 30 ml of 2X NPB from aliquots of the stock solutions stored frozen: 500 mM sucrose (1.5 M stock), 30 mM HEPES (1M stock; titrate with KOH so that pH 7.7 is at 15 mM), 1 mM spermidine trihydrochloride (Sigma S-2501; 10 mM stock), 0.4 mM spermine tetrahydrochloride (Sigma S-1141; 10 mM stock), 2 mM dithiothreitol (Sigma D-0632; 100 mM stock), 2 mM EDTA (500 mM EDTA, pH 8.0).
4. Use the 2XNPB to make a. 30ml 1X NPB, b. 10ml 1XNPB+3%BSA (fraction V, Sigma A-7906), c. 5ml 1XNPB+0.3%BSA.
5. Lysolecithin: 100 μl of 10 mg/ml L-α-lyso-Lecithin, Egg Yolk (Calbiochem, 440154); dissolve at room temperature just before use. Store solid stock at 20°C. Discard the stock powder if it becomes sticky.
6. Bovine serum albumin (BSA): 10% (w/v) BSA (fraction V, Sigma A-7906) Make up 5 ml in water on the day of the sperm nuclei preparation.
7. Sperm storage buffer (1ml) 1X NPB, 30% glycerol, 0.3% BSA.
8. Sperm dilution buffer: 250 mM sucrose, 75 mM KCl, 0.5 mM spermidine trihydrochloride, 0.2 mM spermine tetrahydrochloride. Titrate to pH 7.3-7.5 and store 0.5-1 ml aliquots at 20°C.
9. Hoechst No. 33342 (Sigma B-2261): 10 mg/ml stock in dH₂O, store in a light tight vessel at 20°C.

Equipment:

• Swinging bucket rotor and centrifuge
• cheesecloth
• dissection tools (forceps and scissors)
• fluorescence microscope
• funnel
• gloves
• hemocytometer
• needles (26 gauge)
• paper towels
• petri dishes (60 mm)
• pipettes
• plastic (5 and 10 ml)
• Pipetman tips (1 ml and 200μl)
• Syringes (1 ml)
• tubes (14 ml; Falcon, 2059)
• tubes
• microcentrifuge (1.5 ml)

B. Preparation of High Speed Extract

Reagents:

1. 1X Marc’s Modified Ringer (MMR): 100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM HEPES, pH 7.5. Prepare a 10X stock, and adjust pH with NaOH to 7.5.
2. 20X Extract buffer (XB) salt stock: 2 M KCl, 20 mM MgCl₂, 2 mM CaCl₂, filter-sterilize and store at 4°C.
3. Extract buffer (XB; freshly prepared and stored on ice): 1X XB salts, 50 mM sucrose,10mM HEPES (1 M stock, titrated with KOH so that pH is 7.7 when diluted to 15 mM; filter-sterilize, and store in aliquots at 20°C). Prepare about 100 ml.
4. 2% (w/v) L-Cysteine hydrochloride 1-hydrate: Made up in 1X XB salts before use and titrated to pH 7.8 with NaOH. Prepare about 300 ml.
5. CSF-XB: 1X XB salts, 1 mM MgCl₂ (in addition to MgCl₂ present in XB salts; final concentration 2 mM), 10 mM HEPES, pH 7.7, 50 mM sucrose, 5 mM EGTA, pH 7.7. Prepare 50 ml.
6. Protease inhibitors: Mixture of leupeptin, chymostatin, and pepstatin, each dissolved to a final concentration of 10 mg/ml in dimethyl sulfoxide (DMSO). Store in small aliquots at 20°C.
7. 1 M CaCl₂.
8. Energy mix: 150 mM creatine phosphate, 20 mM ATP, 20 mM MgCl₂.
9. Pregnant Mare Serum Gonadotropin (PMSG): 100 U/ml PMSG (P.G.600®, Intervet, Inc., 021825). Dissolve in water and stored at 20°C.
10. Human Chorionic Gonadotropin (HCG): 1000 U/ml HCG (CHORULON®, Intervet, Inc., 057176 ). Dissolve in water and stored at 4°C.

Equipment:

• Xenopus laevis females
• Needles (18 and 26 gauge)
• Pasteur pipette
• wide bore
• Syringes (1 mL)
• Tubes, microcentrifuge (0.5 mL)
• Tubes, thick-wall polycarbonate (Beckman, 349622)
• Tubes, ultraclear (14 x 95 mm; Beckman, 344060)
• Ultracentrifuge and rotors (e.g., Beckman TL-100 with rotors SW 40 Ti and TLA-100.3)
• Beakers for egg collection
• Buckets or containers for holding female frogs (e.g., 4-L plastic beakers with mesh lids).

C. Nuclear transplantation.

Reagents:

1. 2.5% agarose in 0.1XMMR (for making injection dishes)
2. 2.5% Cysteine in 1XMMR, pH8.0, prepared on the day of use
3. Ficoll
4. 10 mg/ml gentamycin (1000X stock)
5. high speed egg extract (see above)
6. 100 MgCl₂
7. 10X MMR (see above)
8. Restriction enzyme (e.g. NotI from New England Biolabs)
9. Sperm dilution buffer (SDB; see above) and sperm nuclei (see above)
10. Human Chorionic Gonadotropin (HCG) as above
11. mineral oil (Sigma, M8410)
12. Linearized plasmid to be introduced as the transgene: Prepare linearized plasmid at a concentration of about 100 ng/μl in sterile, nuclease-free water (we avoid Tris and EDTA-containing buffers, which are somewhat toxic to embryos). The restriction enzyme used to linearize the plasmid does not have to be the same as the one used in the nuclear transfer reaction. We usually use NotI for all reactions, regardless of what plasmid is linearized with. Some calibration of the enzyme dilution used in the reaction may be necessary, as too much enzyme can cause adverse effects on post-gastrula development. Plasmid can be purified in several different ways: we usually use the Qiagen Qiaquick PCR purification kit according to the manufacturers directions; purification of a single band from a gel is not necessary. If plasmid is purified using phenol/chloroform extractions and ethanol precipitation, be certain to remove all traces of organics and ethanol.

Equipment:

Agarose dishes for injection: In a 60 mm plastic petri dish, lay a small 35mmX35mm weigh boat on molten 2.5% agarose in water 0.1XMMR to create a depression with an agarose-coated bottom for filling with eggs. Once agarose has hardened, wrap in parafilm and store at 4°C until use. Make 2-3 dishes in advance for each transgenic reaction you plan to do.

Infusion pump: We use a single syringe infusion pump from Harvard Apparatus, equipped with a 3 cc syringe/needle filled with mineral oil (Sigma M-8410). Blunt the syringe needle tip (to keep it from perforating the tubing) and attach the fine tygon tubing. Run the pump at ~10nl/sec; this assumes that the time the needle is in each egg will be no greater than 1 sec. Pump should be pre-run for several minutes prior to starting transgenesis for the day to assure that the plunger for the syringe is flush with the piston and that steady positive flow of oil out of the tubing is occurring.

Needles for nuclear transfers. Using a micropipette puller, generate needles with long, sloping tips. Clip these with a forcep under a dissecting microscope equipped with an ocular micrometer to obtain an ~80 micron opening with a beveled shape.

Other equipment: Xenopus laevis females, stereomicroscope, incubator, micromanipulator, microinjection needle puller (e.g. Model P-87, Sutter), syringe needles (26 gauge), glass microinjection needles, ocular micrometer for calibrated clipping of microinjection needle tips to 80μm diameter, petri dishes, weigh boats 35mm, Tygon tubing (ID=1/32 in., OD=3/32 in.)