如何准备转染所需的质粒DNA
转染效率受到诸多因素的影响，除了细胞、培养基和载体等影响因素外，另外一项非常重要的因素便是DNA的质量。为了比较不同厂家的转染效率，我们分别从三家不同的供应商购买了质粒制备试剂盒来制备pEGFP-N3质粒DNA，并通过不同的方法将制备的pEGFP-N3质粒转运至NIH-3T3细胞。 pEGFP-N3质粒分别通过Endofree Maxi Piasmid Kit(Qiagen),PerfectPrep Endofree Piasmid Maxi Kit(5 PRIME,inc)及NucleoBond DNA Maxi Kit(MACHEREY-NAGEL GmbH&Co.KG)制备，我们测定了230,260,280及320nm的吸光值检测DNA的质量。通过上述三种方法制备的DNA质量如下：MACHEREY-NAGEL ＞5Prime＞Qiagen。
pEGFP-N3由PolyJetDNA转染试剂运至NIH-3T3细胞。转染效率的高低与DNA的质量相符，由MACHEREY-NAGEL纯化DNA对应的转染效率最高，而由Qiagen纯化DNA对应的转染效率最低。因此，MACHEREY-NAGEL GmbH & Co.KG的质粒制备试剂盒被认为是制备转染级别DNA的最佳选择。
How to prepare transfection grade plasmid DNA
Transfection efficiencies are affected by a variety of different parameters. Besides factor such as cell culture, medium and vectors, one of most critical parameters is DNA quality. We prepared pEGF-N3 plasmid DNA by large plasmid preparation kits from three different vendors and tested transfection efficiencies by delivering pEGFP-N3 DNAs prepared by different methods to NIH-3T3 cells.
We prepared pEGFP-N3 plasmid with Endofree Maxi Plasmid Kit (Qiagen), PerfectPrep Endofree Plasmid Maxi Kit (5 PRIME, Inc.) and NucleoBond DNA Maxi Kit (MACHEREY-NAGEL GmbH & Co. KG). The DNA quality was determined by measuring absorbance at 230, 260, 280 and 320 nm by spectrometry.
The purity of DNA prepared by the three methods is as follows: MACHEREY-NAGEL > 5 Prime > Qiagen.
Then pEGFP DNA was delivered with PolyJet™ (Cat # SL100688) DNA transfection reagent to NIH-3T3 cells per manufacturer’s protocol. In accordance with DNA purity results, we found that DNA from MACHEREY-NAGEL gave the best transfection efficiency while Qiagen DNA gave the worst efficiency. Therefore plasmid preparation kit from MACHEREY-NAGEL GmbH & Co. KG is confirmed to be the best choice to prepare tansfection grade DNA.
表皮细胞的转染
表皮细胞广泛遍布于身体，正常的表皮细胞较难转染，尤其是使用基于脂质体技术的转染试剂。我们使用电转(Amaxa)方法转染正常人的结肠表皮细胞并得到了65%的GFP标记细胞。非常感谢SignaGen，现在我们使用GenJet Ver II可以成功转染正常人的结肠表皮细胞并且转染效率显著提高至75%。
如下是使用GenJet Ver II(Cat#SL100489)转染表皮细胞的简单步骤： 1、转染时确保表皮细胞达到85%的融合度，并且保证细胞新鲜、健康。
2、对于6孔板，用不含血清的DMEM分别稀释1.0μg,DNA及3.0μl,GenJet Ver.II（Cat＝SL100489）.将稀释好的转染试剂加入DNA中，室温下放置15分钟以形成转染复合物。
3、将转染复合物直接加入表层细胞中：6孔板，每孔含1.0ml培养基，在血清/抗生素存在下，转染进行。
4、在转染进行5小时后，清除转染复合物并换成正常生长培养基。
5、转染后的24-48小时，检测转染基因的表达情况。
中华医学会Transfection of epithelial cells.
Epithelial cells are found throughout the body, from skin to glandular formations within tissues. In vivo these cells are attached to a three dimensional basement membrane matrix. Normal epithelial cell is extremely hard to transfect especially to liposome based transfection reagents. We ever used electroporation (Amaxa) to transfect normal human colonic epithelial cells and got 65% GFP+ cells. Thanks to SignaGen, now we have successfully transfected normal human colonic epithelial cell with GenJet Ver. II with up to 75% GFP+ efficiency. The following is a brief protocol for transfecting epithelial cell with GenJet Ver. II (Cat # SL100489): 1. Grow epithelial cell ~85% confluency at time of transfection. Epithelial cells must freshly prepared and healthy.
2. For 6-well plate, dilute 1.0 μg of DNA and
3.0 μl of GenJet Ver. II (Cat # SL100489) per well with serum free DMEM respectively. Add diluted reagent to diluted DNA and let transfection complex formed at RT for 15 minutes.
3. Add transfection complex to epithelial cells directly. Transfection is conducted in presence of serum/antibiotics with transfection volume of 1.0 ml per well of 6-well plate.
4. Remove transfection complex 5 hours after transfection and change back to normal growth medium.
5. Check transgene expression 24~48 hours post transfection.
针对特定细胞选择更有效的实验步骤
GenJet Ver.II，LipoD293及PolyJet针对哺乳动物细胞的DNA体外转染试剂，均为您提供两种不同的转染步骤——一般步骤及高级步骤，这两种步骤分别针对
不同的哺乳动物细胞。高级步骤主要针对难转的哺乳动物细胞，例如：MDCK，MDA-MB231，Caco-2等细胞。使用一般的转染步骤若是转染效率低于5%，那么使用高级转染步骤则可以将转染效率提升至60%。
如何选择转染步骤则取决于您的细胞特性。对于诸如HEK293，Hela，CHO，3T3，COS，HepG2，LNCaP，PC3，PC12，U2-OS，L929，MCF-7及Huh-7等常用细胞，一般的实验步骤就能得到满意的转染结果。对于诸如MDCK，MDA-MB231，Caco-2，SaoS-2及HUVEC等难转细胞，可以选用高级转染步骤。针对您的细胞，若是您尚不明确选择哪种转染步骤，我们建议您可先尝试一般步骤，如果您使用一般步骤得到的转染效率低于5%，那么您的细胞比较难转，您可尝试高级转染步骤。Choose a more efficient protocol for your specific cell.
GenJet Ver. II, LipoD293 and PolyJet DNA in vitro transfection reagent all offer two protocols for transfecting mammalian cells--------a general protocol and an advanced protocol. The two protocol are written for transfecting different types of mammalian cells. The advanced protocol which involves a shaving cell technology is for very hard to transfect mammalian cells like MDCK, MDA-MB231, Caco-2, etc. The general protocol usually gives less than 5% efficiency while advanced protocol can get up to 60% efficiency on these problematic cells.
The principle to decide which one protocol is used to your cell is highly dependent upon the nature of your cell. For commonly used cells like HEK293, Hela, CHO, 3T3, COS, HepG2, LNCaP, PC3, PC12, U2-OS, L929, MCF-7 and Huh-7, be sure to use general protocol which usually gives very good transgene expression while the advanced protocol does not help. For very hard to transfect cells like MDCK, MDA-MB231, Caco-2, SaoS-2 and HUVEC, follow the advanced protocol by trypsinizing these adherent cells first followed by incubation of the cell pellet with transfection complex. If you do not know for your specific cells how to choose the more efficient protocol, we suggest you try general protocol first. If you get less than 5% efficiency with the general protocol, then your cell is hard to transfect in nature and you can consider trying advanced one.
如何优化PolyJet转染试剂
我们使用PolyJet DNA转染试剂转染表皮细胞及Raw267.4细胞非常有效，并且毒性很小。对于如何更好的优化PolyJet转染试剂，我乐意分享以下几点：
1、DNA的质与量。DNA的纯度对于转染实验至关重要。由E Coli制备的DNA，
A260/280必须达到1.80甚至更高。对于6孔板，通常每孔~1.0μg DNA足矣。其他规格的细胞培养皿，可以根据表面积适当调整DNA含量。
2、PolyJet/DNA的比率。对于表皮及Raw267.4细胞的转染，我们将PolyJet/DNA 的比率固定在3:1，并且得到了满意的结果，没有为寻更合适的比率问题而烦恼。
3、稀释。DNA及PolyJet的稀释一定不要含有血清。我们使用的是不含血清的高糖DMEM培养基，效果很好。请不要选择Opti-MEM，因为它可以影响转染复合
物的形成，因此，千万不要使用Opti-MEM来稀释质粒及PolyJet。
4、转染中可以含有血清。我们在含有血清/抗生素及不含二者的情况下分别进行可转染实验，没有发现两种情况下转染效率有什么差别。因此，血清/抗生素对PolyJet转染试剂没有什么影响。
知觉现象学>阿继电器Some technical tips for optimizing PolyJet reagent.
PolyJet DNA Transfection Reagent is very efficient to transfect epithelial and Raw 267.4 cells in our hands without significant toxicity. Here I would like to share several technical points for better using PolyJet transfection reagent.
1. DNA amount and quality. DNA purity is essential for high efficiency. DNA prepared from E Coli. must be 1.80 at A260/280 or higher. For 6-well plate, ~1.0 μg of DNA per well is usually good enough. For other cell culture format, just adjust DNA amount per culture dish's surface area.
2. Ratio of PolyJet/DNA. For epithelial and Raw 267.4 cells, we locked the ratio at 3:1 with excellent results and never bothered to find the optimal ratio.
3. Diluent. Diluent used to dilute DNA and PolyJet reagent must be serum free. Serum-free DMEM medium with high glucose works for us. Opti-MEM is NOT a good choice because it may affect formation of transfection complex. So never use Opti-MEM as dilute.
4. Transfection with serum. We performed transfection with or without serum/antibiotics and failed to find any difference in efficiency. Therefore, PolyJet reagent is NOT interfered by serum/antibiotics.
如何优化LipoD293转染试剂
LipoD293DNA转染试剂是脂质体DNA转运工具的升级版本。我们实验室使用LipoD293DNA转染试剂成功转染了HepG2，LNCaP，CHO及HEK293细胞。
接下来，我们乐意就如何提高转染效率，降低毒性等方面的小技巧同大家分享。
1、LipoD293/DNA的比率。尽管合适的比率由细胞类型决定，但是使用LipoD293/DNA，3:1的固定比率通常能得到很好的转染效率。
2、每孔中DNA的含量。对于24孔板来说，我每孔使用的0.2到0.5μgDNA。太多的DNA（例如每孔1.0μg）没有必要，并且会产生较高的毒性。其他规格的细胞培养器皿，可以根据表面积适当调整DNA含量。
3、DNA及LipoD293的稀释。一定不要使用含有血清的培养基来稀释二者。高糖的Plain DMEM培养基是不错的选择，但是，高糖并不是很重要。千万不要使用Opti-MEM（invitrogen生产）！Opti-MEM可以影响转染复合物的形成。我的同事没有认识到该点的重要性，错误地使用了Opti-MEM，导致转染效率低下。
4、血清/抗生素的存在与否对转染没有影响。目前，我们正在使用的哺乳动物细胞通常是用LipoD293进行转染的，血清/抗清素对于转染效率没有影响。与其他依赖于脂质体的转染试剂不同，LipoD293不受血清/抗生素的影响，因此您不必担忧转染中含血清培养造成的高细胞死亡率问题。
5、转染后更换培养基，对于我们正在研究的哺乳动物细胞，通常，我们是在转
染后24小时更换培养基，没有必要在转染后5小时更换培养基。
Some points which are important but ofetn neglected for optimizing LipoD293 reagent.
LipoD293 DNA Transfection Reagent is an enhanced version of liposome DNA delivery tool. Our lab has been using LipoD293 DNA transfection reagent for transfecting HepG2, LNCaP, CHO and HEK293 cells with very good lucks. Now we like to share some technical points which I think are important for maximum efficiency and minimal toxicity and often ignored:
1. Ratio of LipoD293 reagent/DNA. While the optimal ratio is cell type dependent, the ratio of reagent/DNA locked at 3:1 often gives excellent efficiency. Our labs has been transfecting HepG2, LNCaP, CHO and HEK293 with LipoD293 reagent at 3:1 ratio with very good efficiency.
2. DNA amount per well. For 24-well plate, I used 0.2 to 0.5 μg of DNA per well. Too much DNA (eg, 1.0 μg per well) is unnecessary and might lead to high cytotoxicity. For other cell culture format, adjust DNA amount per culture dish's surface area.
空间贴图3. Diluent for diluting DNA and LipoD293 reagent. Diluent must be serum free. Plain DMEM medium
with high glucose is usually very good. High glucose seems not that important. Never use Opti-MEM (from Invitrogen)! This is very important 'cause my colleagues often failed to recognize importance of the diluent and misused Opti-MEM, leading to suboptimal efficiency. Per our initial test, Opti-MEM is able to disrupt formation of transfection complex.
4. Transfection with or without serum/antibiotics. For all mammalian cells we are currently working on, we always perform transfection with LipoD293 reagent in the presence of serum (10% FBS)/antibiotics without compromising any efficiency, thus greatly simplifying our transfection procedures. Unlike other liposome based reagent, LipoD293 reagent is usually NOT interfered by serum/antibiotics, so do not bother to perform transfection in serum-free medium which otherwise results in high cell death.
5. Medium change post transfection. We usually change medium 24 hours after transfection. Medium change 5 hours post transfection is not necessary for all the mammalian cell we are currency using.
转染L929细胞的简单步骤
L929是小鼠成纤维细胞瘤细胞株，经常被用来检测TNF-alpha及TNF-beta。对TNF的处理经常会引发
细胞凋亡及死亡，因此L929细胞经常被用作免疫分析。但是，转染L929细胞非常难，尤其使用基于脂质体技术的转染试剂，我们使用GenJet VerⅡ及PolyJet转染L929细胞获得了75%的转染效率，简单的实验步骤如下。
1、转染时确保L929细胞达到80%的融合度，细胞必须健康并且传代不能超过9国际学术会议
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