文章詳情
體內轉染技術要點
日期：2024-12-28 06:29
瀏覽次數：529
摘要：
1. 質粒的制備
體內轉染對質粒的要求更嚴格，建議使用高質量的質粒，無RNA污染，無內**和低蛋白成分。質粒的純度低會嚴重影響轉染的效率，雜質和內**的存在會造成一定的**原性和毒性，嚴重者造成動物和人的死亡。
2. 葡萄糖稀釋液
我們推薦使用無菌的等離子的10% 葡萄糖溶液（W/V）制備in vivo jetPEI/DNA復合物（終濃度為5% 的葡萄糖溶液），因為在無鹽或低鹽的條件下，才能形成穩定的小顆粒復合物。
3. 以小鼠為例，介紹不同注射途徑時的轉染...
1. 質粒的制備
體內轉染對質粒的要求更嚴格，建議使用高質量的質粒，無RNA污染，無內**和低蛋白成分。質粒的純度低會嚴重影響轉染的效率，雜質和內**的存在會造成一定的**原性和毒性，嚴重者造成動物和人的死亡。
2. 葡萄糖稀釋液
我們推薦使用無菌的等離子的10% 葡萄糖溶液（W/V）制備in vivo jetPEI/DNA復合物（終濃度為5% 的葡萄糖溶液），因為在無鹽或低鹽的條件下，才能形成穩定的小顆粒復合物。
3. 以小鼠為例，介紹不同注射途徑時的轉染情況。
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Tail vein injection
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DNA: 50 μg
in vivo-jetPEI?: 5-10 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 ? inch
26 gauge needle and a 1 ml syringe.
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Intracerebral injection (stereotaxic injection)
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DNA: 1 μg (for 8-12 week-old mice)
in vivo-jetPEI?: 0.12 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).
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Retro-orbital injection
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DNA: 40 μg
in vivo-jetPEI?: 6.4 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.
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Nasal instillation for trachea
and lung delivery
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DNA: 20 μg
in vivo-jetPEI?: 2-4 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.
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Intraperitoneal injection
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DNA: 100 μg
in vivo-jetPEI?: 16-20 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 ?
inch 26 gauge needle and a 1 ml
syringe.
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Intratumoral injection
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DNA: 10-20 μg
in vivo-jetPEI?: 1-4 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.
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