【讨论】RNAi与慢病毒载体
丁香园论坛
7399
近年来的研究表明，将与mRNA对应的正义RNA和反义RNA组成的双链RNA(dsRNA)导入细胞，可以使mRNA发生特异性的降解，导致其相应的基因沉默。这种转录后基因沉默机制(PTGS)被称为RNA干扰（RNAi）。
通过构建RNAi表达载体制备shRNA(发卡结构小干扰RNA)，已成为进行RNA干扰实验的常用手段之一。 而目前为了感染原代细胞和难感染的细胞系或者在动物水平进行RNAi，研究科学家更多的选择了慢病毒载体。在RNAi的研究中，为了感染原代细胞和难感染的细胞系，科学家开始借助于病毒载体实现RNAi；由于慢病毒可以同时感染分裂和非分裂细胞、整合性以及免疫原性低等优点，目前在RNAi的研究中，基于慢病毒构建的shRNA被最广泛的使用。而在RNAi研究需要长期观察或者需要进行动物实验时，基于慢病毒构建的shRNA的优势更为明显。
慢病毒的应用前景相当广。。。
通过构建RNAi表达载体制备shRNA(发卡结构小干扰RNA)，已成为进行RNA干扰实验的常用手段之一。 而目前为了感染原代细胞和难感染的细胞系或者在动物水平进行RNAi，研究科学家更多的选择了慢病毒载体。在RNAi的研究中，为了感染原代细胞和难感染的细胞系，科学家开始借助于病毒载体实现RNAi；由于慢病毒可以同时感染分裂和非分裂细胞、整合性以及免疫原性低等优点，目前在RNAi的研究中，基于慢病毒构建的shRNA被最广泛的使用。而在RNAi研究需要长期观察或者需要进行动物实验时，基于慢病毒构建的shRNA的优势更为明显。
慢病毒的应用前景相当广。。。
请问慢病毒包装必须用293T细胞吗?
回复ddandgg： 慢病毒包装时除了要用慢病毒质粒，包装质粒及衣壳质粒外 还必须借助宿主细胞产生的蛋白形成病毒外壳。293T细胞表达E1A基因，后者能激活一些病毒启动子进而产生大量包装病毒所需要的蛋白。
非常感谢jiachengyou2007的回复！请问293A和293T有什么区别？293A细胞不表达E1A基因吗？
293A中A的意思是adherent，293T中T的意思是S40 large T antigen(大T抗原)。293A细胞是293来源的细胞，含有腺病毒E1早期基因，而我们使用的腺病毒载体为了提供克隆的空间，即装载外源基因空间，缺失了E1的早期区，因此当我们转然Ｅ１缺失的病毒载体到293A时，由于293A能提供E1早期蛋白，才能形成有感染力的腺病毒颗粒。腺病毒的包装使用的是293A细胞。另外，293T也并不是专门用来包装慢病毒的，还可以有许多其它用途。293T可以包装aa,三质粒共转染的方法里，对293细胞的要求就是细胞要表达腺病毒的E1A,E1B蛋白，即腺病毒的E1A和E1B基因已经插入了293细胞，293T即满足这样的要求，所以它可以用于三质粒共转包装病毒
谢谢gjq8523详细的解释！你有没有关于293细胞的详细资料让我共享一下啊？我还有一个疑问，按照你的解释是不是293A和293T都可以用于腺病毒和慢病毒的包装啊？可是我看到的所有文献中慢病毒都是用293T包装的，如何解释？换句话说，用293A包装慢病毒是否可行？
以下信息来自zhujoker The 293T/17 cell line is a derivative of the 293T (293tsA1609neo) cell line. 293T is a highly transfectable derivative of the 293 cell line into which the temperature sensitive gene for SV40 T-antigen was inserted. 293T cells were cloned and the clones tested with the pBND and pZAP vectors to obtain a line capable of producing high titers of infectious retrovirus, 293T/17. These cells constitutively express the simian virus 40 (SV40) large T antigen, and clone 17 was selected specifically for its high transfectability.293T/17 cells were cotransfected with the pCRIPenv- and the pCRIPgag-2 vectors to obtain the ANJOU 65 (see ATCC CRL-11269) cell line.ANJOU 65 cells were cotransfected with the pCRIPgag-2 and pGPT2E vectors to obtain the BOSC 23 (see ATCC CRL-11270) ecotropic envelope-expression packaging cell line.ANJOU 65 cells were also cotransfected with the pCRIPAMgag vector along with a plasmid expressing the gpt resistance gene to obtain the Bing (see ATCC CRL-11554) amphotropic envelope-expression packaging cell line.
293细胞是转染腺病毒E1A基因的人肾上皮细胞系，293T细胞由293细胞派生，同时表达SV40大T抗原，含有SV40复制起始点与启动子区的质粒可以复制。用Ca3(PO4)2转染效率可高达50%。蛋白表达水平高，转染后2-3天用碱性磷酸酶分析可较容易地检测到表达的蛋白。瞬时转染293T细胞是过表达蛋白并获得细胞内及细胞外（分泌的或膜）蛋白的便捷方式。293细胞比较容易转染，是一个很常用的表达研究外源基因的细胞株。293细胞还有一个衍生株：293T/17的转染效率更高，成为广大研究者研究基因功能的一个强大工具。
293细胞是转染腺病毒E1A基因的人肾上皮细胞系，293T细胞由293细胞派生，同时表达SV40大T抗原，含有SV40复制起始点与启动子区的质粒可以复制。用Ca3(PO4)2转染效率可高达50%。蛋白表达水平高，转染后2-3天用碱性磷酸酶分析可较容易地检测到表达的蛋白。瞬时转染293T细胞是过表达蛋白并获得细胞内及细胞外（分泌的或膜）蛋白的便捷方式。293细胞比较容易转染，是一个很常用的表达研究外源基因的细胞株。293细胞还有一个衍生株：293T/17的转染效率更高，成为广大研究者研究基因功能的一个强大工具。
基本上都是用293T细胞来包装腺病毒或慢病毒 293A 与293T 的详细区别本人还真的不很清楚，什么时候查到了，一定早日分享与你。
刚看到篇文献，用的是慢病毒载体感染MDCK来获得shRNA
路过学习了，继续关注ing
非常感谢！
Lentiweb Protocols
Protocol for Production of Lentiviral Vectors in 293T cells
Day 1 Plating (9-10am)
Plate 2-2.5x106 of 293T cells per 10cm plate
Day 2 Transfection (9-10am)
Prepare calcium-phosphate precipitate (1ml/10cm plate)
Transfer vector - 20µg
Packaging plasmid - 15µg (3rd generation: pMDL g/p RRE - 10µg + pRSV-Rev - 5µg)
Envelope plasmid - 6µg
Add water to 0.5ml, add 0.5ml 2xHBS and mix well. Add 50µl 2.5M CaCl2 and shake briefly, keep in RT for 20-25min, add dropwise on a plate and mix gently with a medium
Change medium (6-8hrs later); remove medium with precipitate and add 6ml/plate of fresh medium
Day 4 Collection (9-10am)
Collect medium
Spin 3000rpm/5min/RT
Filter through 0.45 µm
At this point virus can be used for transduction, frozen at -70°C for future use, or concentrated
Concentration
Transfer 30ml of virus to 33ml Beckman conical tubes spin at 26.000rpm/2hrs/4°C in Beckman SW28 swingle bucket rotor. After spin discard supernatant and resuspend the virus in a desired volume of serum-free medium (e.g. Cellgro or Episerf) or PBS/1% BSA, aliquot and store at -70°C. For transduction of very delicate cells the virus can be concentrated on sucrose cushion, just put 4ml of 20% sucrose on the bottom of the tube and overlay with 26ml of viral supernatant.
Reagents:
2 x HBS (for 500ml)
NaCl - 8g
KCl - 0.38g
Na2HPO4 - 0.1g
Hepes - 5g
Glucose - 1g Bring pH to 7.05
2.5M CaCl2
bi-distilled water
siRNA Design
1. Select siRNA 19-mers
First, look around in the literature if any siRNA for your gene has been already made or have a look at siRNA Database (small though)
There is a growing numbers of tools for selection of siRNA 19-mers based on a provided cDNA sequence. We use siRNA Selection Program devoloped at Whitehead Institute. You need to register (for free) in order to obtain username and password. I usually go for custom AAN19 (AA is present only in the target sequence and is not a part of your siRNA). Recently, a very nice software - iRNAi - was developed by MEK&Tosj from Amsterdam. iRNAi searches for oligos, does thermodynamic profile and outputs the oligo sequences in ready to order hairpin design (also fully customizable), and many more. It works great, it's free and only for MacOSX :).
It has been reported that effective RNAi depends on siRNA-specifc properties, rather then properties of target mRNA and the most potent siRNA are characterized by:
moderate to low G/C content 36-52%
low internal stability of the sense 3'end; at least one A/T duplex between position 15-19
lack of internal repeats; Tm < 60 °C
A at position 3, U at position 10, G at position 13, A at position 19 (sense strand) and absence of G at position 19
In order to minimize off-target effects use oligos with at least 3 mismatches to unrelated sequences. Please see Reynolds et al, Nat Biotech 22:326-30, 2004 for more details.
2. Fine tuning - thermodynamics
It has been reported that enhanced flexibility at 5' anti-sense terminal base pair play critical role in siRNA function. In another words, have e.g. G-C pair at pos. 1 (that is 5' sense end) and A-T or even a A-G mismatch at position 19 (that is 5' antisense) of your siRNA sequence. However, we have not tested this concept in our lab yet and (e.g. if that flexibility would not affect hairpin structure). But the data looks very nice and the strategy is worth to try. In case you did tested this strategy and have comments or suggetstions to share, please send me an email and I will post it here. Please see D. Schwarz et al, Cell 115:199, 2003 and A. Khvorova et al, ibid for details.
3. Design a hairpin
Avoid polyT (4 and more) stretches since it may lead to premature termination of shRNA transcription. Also, make sure that your N19 does not end with 2-3Ts; since the hairpin loop starts already with 2Ts. We order most of our oligos from Sigma at 0.05µmol range. Please consult our Cloning Strategies for details. More detailed informations can be found e.g. at T. Tuschl Website and Ambion, specially their siRNA design guidelines
4. Clone hairpin into a vector
Please consult our Cloning Strategies or recent publication for informations how to design expression vector with siRNA (shRNA).
Protocol for Production of Lentiviral Vectors in 293T cells
Day 1 Plating (9-10am)
Plate 2-2.5x106 of 293T cells per 10cm plate
Day 2 Transfection (9-10am)
Prepare calcium-phosphate precipitate (1ml/10cm plate)
Transfer vector - 20µg
Packaging plasmid - 15µg (3rd generation: pMDL g/p RRE - 10µg + pRSV-Rev - 5µg)
Envelope plasmid - 6µg
Add water to 0.5ml, add 0.5ml 2xHBS and mix well. Add 50µl 2.5M CaCl2 and shake briefly, keep in RT for 20-25min, add dropwise on a plate and mix gently with a medium
Change medium (6-8hrs later); remove medium with precipitate and add 6ml/plate of fresh medium
Day 4 Collection (9-10am)
Collect medium
Spin 3000rpm/5min/RT
Filter through 0.45 µm
At this point virus can be used for transduction, frozen at -70°C for future use, or concentrated
Concentration
Transfer 30ml of virus to 33ml Beckman conical tubes spin at 26.000rpm/2hrs/4°C in Beckman SW28 swingle bucket rotor. After spin discard supernatant and resuspend the virus in a desired volume of serum-free medium (e.g. Cellgro or Episerf) or PBS/1% BSA, aliquot and store at -70°C. For transduction of very delicate cells the virus can be concentrated on sucrose cushion, just put 4ml of 20% sucrose on the bottom of the tube and overlay with 26ml of viral supernatant.
Reagents:
2 x HBS (for 500ml)
NaCl - 8g
KCl - 0.38g
Na2HPO4 - 0.1g
Hepes - 5g
Glucose - 1g Bring pH to 7.05
2.5M CaCl2
bi-distilled water
siRNA Design
1. Select siRNA 19-mers
First, look around in the literature if any siRNA for your gene has been already made or have a look at siRNA Database (small though)
There is a growing numbers of tools for selection of siRNA 19-mers based on a provided cDNA sequence. We use siRNA Selection Program devoloped at Whitehead Institute. You need to register (for free) in order to obtain username and password. I usually go for custom AAN19 (AA is present only in the target sequence and is not a part of your siRNA). Recently, a very nice software - iRNAi - was developed by MEK&Tosj from Amsterdam. iRNAi searches for oligos, does thermodynamic profile and outputs the oligo sequences in ready to order hairpin design (also fully customizable), and many more. It works great, it's free and only for MacOSX :).
It has been reported that effective RNAi depends on siRNA-specifc properties, rather then properties of target mRNA and the most potent siRNA are characterized by:
moderate to low G/C content 36-52%
low internal stability of the sense 3'end; at least one A/T duplex between position 15-19
lack of internal repeats; Tm < 60 °C
A at position 3, U at position 10, G at position 13, A at position 19 (sense strand) and absence of G at position 19
In order to minimize off-target effects use oligos with at least 3 mismatches to unrelated sequences. Please see Reynolds et al, Nat Biotech 22:326-30, 2004 for more details.
2. Fine tuning - thermodynamics
It has been reported that enhanced flexibility at 5' anti-sense terminal base pair play critical role in siRNA function. In another words, have e.g. G-C pair at pos. 1 (that is 5' sense end) and A-T or even a A-G mismatch at position 19 (that is 5' antisense) of your siRNA sequence. However, we have not tested this concept in our lab yet and (e.g. if that flexibility would not affect hairpin structure). But the data looks very nice and the strategy is worth to try. In case you did tested this strategy and have comments or suggetstions to share, please send me an email and I will post it here. Please see D. Schwarz et al, Cell 115:199, 2003 and A. Khvorova et al, ibid for details.
3. Design a hairpin
Avoid polyT (4 and more) stretches since it may lead to premature termination of shRNA transcription. Also, make sure that your N19 does not end with 2-3Ts; since the hairpin loop starts already with 2Ts. We order most of our oligos from Sigma at 0.05µmol range. Please consult our Cloning Strategies for details. More detailed informations can be found e.g. at T. Tuschl Website and Ambion, specially their siRNA design guidelines
4. Clone hairpin into a vector
Please consult our Cloning Strategies or recent publication for informations how to design expression vector with siRNA (shRNA).
路过学习了
路过，好好学习，
学习了，谢谢！
受益了
学习一下，挺有用！
我们的慢病毒滴度很好，QQ24289096
求一份cox-2慢病毒包装的protocol，多谢！！
本文由丁香园论坛提供，想了解更多有用的、有意思的前沿资讯以及酷炫的实验方法的你，都可以成为师兄的好伙伴
师兄微信号：shixiongcoming
