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体内转染技术要点

作者:admin 信息来源:达科为 日期:20110817 打印 字体:  
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1.       质粒的制备
体内转染对质粒的要求更严格,建议使用高质量的质粒,无RNA污染,无内毒素和低蛋白成分。质粒的纯度低会严重影响转染的效率,杂质和内毒素的存在会造成一定的免疫原性和毒性,严重者造成动物和人的死亡。
 
2.       葡萄糖稀释液
我们推荐使用无菌的等离子的10% 葡萄糖溶液(W/V)制备in vivo jetPEI/DNA复合物(终浓度为5% 的葡萄糖溶液),因为在无盐或低盐的条件下,才能形成稳定的小颗粒复合物。
 
3.       以小鼠为例,介绍不同注射途径时的转染情况。
Tail vein injection
DNA: 50 μg
in vivo-jetPEI5-10 μl
N/P ratio: 5-10
Injection volume: 200-400 μl, 5% glucose
Method: The mouse is placed in a restainer and 70% ethanol
is applied on the tail in order to slightly swell the vein. Complexes in solution are injected into thetail vein over 10 sec, using a ½ inch
26 gauge needle and a 1 ml syringe.
 
 
Intracerebral injection (stereotaxic injection)
DNA: 1 μg (for 8-12 week-old mice)
in vivo-jetPEI0.12 μl
N/P ratio: 6
Injection volume: 5 μl, 5% glucose
Method: Single injection (5 μl) into either
lateral ventricle (0.2 mm posterior to the
bregma line, 1.1 mm lateral, and 2.2 mm
deep from the pial surface) to pentobarbital
anesthetized mice (65 mg/kg).
 
 
 
Retro-orbital injection
DNA: 40 μg
in vivo-jetPEI6.4 μl
N/P ratio: 8
Injection volume: 200-400 μl, 5% glucose
Method: The tip of a 27 g hypodermic needle is introduced
carefully in front of the eye. Follow the edge of the orbit down until
feeling the needle tip at the base beneath the eye. Inject complexesin solution within 2 sec. If performed carefully, there will be little or
no bleeding. The capillary nexus will take up the injected solution
rapidly.
 
Nasal instillation for trachea
and lung delivery
DNA: 20 μg
in vivo-jetPEI2-4 μl
N/P ratio: 5-10
Injection volume: 50-200 μl, 5% glucose
Method: Mice are held supine at an angle of 45° with pressure applied to the
lower mandibule to immobilize the tongue and prevent swallowing. Complexes
in solution are then introduced to the nasal planum using a micropipet.
 
 
Intraperitoneal injection
DNA: 100 μg
in vivo-jetPEI16-20 μl
N/P ratio: 8-10
Injection volume:
400 μl to 1 ml, 5% glucose
Method: Complexes in solution
are injected into the peritoneal
cavity over 10 sec, using a ½
inch 26 gauge needle and a 1 ml
syringe.
 
 
Intratumoral injection
DNA: 10-20 μg
in vivo-jetPEI1-4 μl
N/P ratio: 5-10
Injection volume: 50-100 μl, 5% glucose
Method: For implanted subcutaneous tumors
(size> 5 mm³), perform multiple injections of
10-20 μl complexes at different sites of the tumor to
avoid reflux.
 
 
Nebulization for trachea and lung delivery
DNA: 40 μg
in vivo-jetPEI4-8 μl
N/P ratio: 5-10
Nebulized volume: 4-10 ml, 5% glucose
Method: Mice are placed unrestained in a whole-body aerosol exposure
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