PEI轉(zhuǎn)染手冊(cè)

轉(zhuǎn)染操作流程：
 
  1、細(xì)胞傳代：

轉(zhuǎn)染前24h內(nèi)分離293T細(xì)胞至含10％胎牛血清的DMEM培養(yǎng)基 中進(jìn)行傳代培養(yǎng)，接種密度如下：
         6孔板：0.5x10⁶細(xì)胞
         10cm培養(yǎng)皿：4.0x106細(xì)胞
         15cm培養(yǎng)皿：9.0x106細(xì)胞

2、轉(zhuǎn)染

轉(zhuǎn)染前將所有試劑置于室溫。

2.1質(zhì)粒DNA的稀釋

在無菌試管中，用無血清的DMEM W / O酚紅培養(yǎng)基（濃度為10％）稀釋質(zhì)粒DNA（ug）。病毒包裝體（psPAX2）：病毒包膜（pMD2G）和重組質(zhì)粒DNA的稀釋比例分別為4:2:1。
       

6孔板：200ul+3ug的DNA
        10cm培養(yǎng)皿：1ml+7-8ug的DNA
        15cm培養(yǎng)皿：2ml+11-12ug的DNA

   2.2 轉(zhuǎn)染復(fù)合體形成

將PEI（1ug/uL）加入已稀釋的總DNA中，PEI與DNA（ug）的混合比率為3:1。加入后立即渦旋混合。
      

 6孔板：9ul PEI復(fù)合體（1ug/ul）=9ug
        10cm培養(yǎng)皿：21ul PEI復(fù)合體（1ug/ul）=21ug
        15cm培養(yǎng)皿：33ul PEI復(fù)合體（1ug/ul）=33ug
將添加到細(xì)胞的DNA/ PEI的混合物在室溫下孵育15分鐘。

4、收獲轉(zhuǎn)染細(xì)胞

轉(zhuǎn)染后48小時(shí)內(nèi)收獲轉(zhuǎn)染細(xì)胞/或病毒上清。
 
試劑的配制：
PEI（1ug/ul） Polysciences（CAT＃23966-2配制成儲(chǔ)液：)
1、將無內(nèi)毒素的無菌水加熱至80℃左右溶解PEI，冷卻到室溫。
2、調(diào)整pH值至7.0，用0.22um的濾器過濾消毒，分裝后儲(chǔ)存在-20℃。工作液可保持在4℃

訂貨信息

友情提醒：
Polyethylenimine, Linear (MW 25,000) 23966-2為Polysciences公司生產(chǎn)的化合物產(chǎn)品，每批產(chǎn)品出廠前都經(jīng)過嚴(yán)格的檢測,保證每批產(chǎn)品都*、穩(wěn)定且高品質(zhì),但由于作為轉(zhuǎn)染試劑使用過程中有很多實(shí)驗(yàn)室因素(PH,水純度,稱量的準(zhǔn)度,dna純度…)造成溶解出現(xiàn)問題或者轉(zhuǎn)染效率出現(xiàn)差異,所以廠家只受理該產(chǎn)品純度，分子量及重量缺失破損等相關(guān)投訴，針對(duì)極個(gè)別客戶溶解問題和轉(zhuǎn)染效率問題我們只能友善解決,所以不能保證您能獲得滿意答復(fù)，對(duì)于產(chǎn)品應(yīng)用方法的問題，不作為評(píng)價(jià)我們售后工作的依據(jù)。
如果我們提供的PEI手冊(cè)都無法解答您的問題,建議您可以多看文獻(xiàn),多調(diào)整一下轉(zhuǎn)染條件,找出適合自己細(xì)胞的轉(zhuǎn)染方法。如果無暇摸索條件,您也可以直接購買我們EZ Trans細(xì)胞轉(zhuǎn)染液,這樣免去您很多煩惱,謝謝理解

改進(jìn)的PEI轉(zhuǎn)染試劑EZ Trans細(xì)胞轉(zhuǎn)染液 96孔細(xì)胞培養(yǎng)板用量  現(xiàn)實(shí)中濃度請(qǐng)調(diào)整，MAX請(qǐng)適當(dāng)調(diào)整濃度

改進(jìn)的PEI轉(zhuǎn)染試劑Sinofection和其他產(chǎn)品的對(duì)比

Transfection with Sinofection has been proved to provide higher levels of protein expression in several cell lines than leading competitor’s products. For large-scale production of recombinant proteins, Sinofection is the best choice to achieve the optimum yield. In addition, Sinofection also exhibits high transfection efficiency to ensure successful transfections.

Fig. 1 Protein expression levels by transient transfection using Sinofection and other competitors were compared in 9 cell lines, 6 of which showed superior results transfected by Sinofection （A~F）. Protein expression level was determined by Elisa assay. The transfection by competitor transfection reagents followed the manufacturers’ protocols.

Excellent transfection of both adherent and suspension cells for a broad range of cell lines

Hundreds of recombinant antibodies and proteins have been successfully expressed by transient or stable transfection with Sinofection at various scales, from 0.1ml in a 96-well plate to 50L in a bioreactor. Utilizing EZ Trans細(xì)胞轉(zhuǎn)染液 for all scales of transfection application will save the cost of research and production to a great extent.

Striking stability at 37°C

Long-term stability test showed Sinofection remained its activity at 37°C for at least 4 months. Storage at -20°C~RT can maintain stable for at least 6 months. Exceptional stability resulted from rigorous screening and reliable investigation for a long time. Thus the stability of Sinofection can outperform any other transfection reagent.

Fig. 2. Stability test for Sinofection at 4 temperatures during 26 weeks. One recombinant antibody and recombinant VEGF-Fc were tested for expression level by transient transfection with Sinofection stored under different conditions. Expression level was assayed by Elisa.

Easy and fast procedure

Transfection protocol （35-mm dish）:

Preparation of cells

• Adherent cells
  • One day before transfection, plate 0.2–2×106 cells in medium per 35-mm dish. Incubate at 37°C （5% CO2） overnight. The confluency of cells should be 50–80% before transfection.
• Suspension cells
  • Plate 2 ml freshly passaged cells at a density of 5 × 104/ml to 1 × 106/ml in a 35-mm dish. The suspension cells should be in log growth phase at the time of transfection. Incubate cells at 37°C （5% CO2） overnight. The cell number depends on your needs and the cell type to be transfected.

Preparation of EZ Trans細(xì)胞轉(zhuǎn)染液&DNA complexes

Dilute DNA with appropriate diluents, such as PBS, NaCl solution, glucose solution, DMEM, medium with or without serum, whichever is isotonic, to a concentration of 20 μg/ml （optimization recommended） and a total volume of 250 μl.

Dilute EZ Trans細(xì)胞轉(zhuǎn)染液 of appropriate amount in 250 μl of medium.

Combine the diluted DNA with the diluted Sinofection （total volume = 500 μl）. Mix gently and incubate for 20 minutes at room temperature （solution may appear cloudy）.

Transfection

Add the 500 μl complexes drop-wise to cells and medium. Gently rock the dishes or wells to ensure even distribution.

Incubate cells at 37°C in a CO2 incubator for 18-48 hours （normally high expression is achieved after 72 hours） prior to protein expression assay.

Transfection protocol （flask）:

Transfecting Cells for Protein ExpressionThis protocol is typically used to transfect DNA into 293 EBNA or CHO cells cultured in flasks. Use sterile DNA or filter-sterilize DNA before use （re-quantify your DNA after filtration）.

All amounts are on a per flask basis for 30 ml cultures in a 125 ml shake flask; for other formats, see Table 2. Scaling up or Down Transfections.

Table 2. Scaling up or down transfections with Sinofection. Note: the actual conditions should be optimized by experiments.

1. Pass 293 cells at 0.3 × 105 cells/ml, shake at 175 rpm. After 72 hours, dilute cells to 1.0× 106 cells/ml, shake at 175 rpm. Transfect after 4 hours.
2. Pass CHO cells at 0.3 × 105 cells/ml, shake at 175 rpm. After 48 hours, dilute cells to 1.0× 106 cells/ml, shake at 175 rpm. Transfect after 4 hours.
3. Dilute the required plasmid DNA amount with appropriate diluent, such as PBS with Mg2+, 150mM NaCl, 300mM glucose, DMEM medium with or without serum, whichever is isotonic. Incubate for 5 min at room temperature. Add the required Sinofection volume to the diluted DNA and mix gently to assure a total volume of 1.5~3ml.
4. Incubate the mixture for 10-20 minutes at room temperature to allow complexes to form. Do not incubate for longer than 20 minutes.
5. Slowly add 1.5~3ml of DNA-lipid mixture into the 125ml flask containing cells while slowly swirling the flask.
6. Incubate transfected cell cultures at 37°C, 8% CO2 on an orbital shaker set to 175 rpm for both 293 cells and CHO cells.

Low cytotoxicity

EZ Trans細(xì)胞轉(zhuǎn)染液 demonstrates very low cytotoxicity which is tested and verified by in-house WST-8 assay for each lot. The WST-8 assay is based on the colorimetric reduction of water soluble tetrazolium （WST） by mitochondrial dehydrogenase

The absorbance of formazan dye solution is in direct proportion to the number of viable cells. Compared with the prev alent WST-1 method, the sensitivity of WST-8 assay is higher meanwhile the solubility and stability of formazan dye is better.

Fig. 3. Microscopy comparison of HeLa cells and DG44 cells transfected with L2K and Sinofection respectively. Transfections were performed following the manufacturer’s recommendations.

Fig. 4. Cytotoxicity comparison of Sinofection and L2K for 4 cell lines. Transfections were performed following the manufacturer’s recommendations.

 常規(guī)轉(zhuǎn)染技術(shù)可分為兩大類，一類是瞬時(shí)轉(zhuǎn)染，一類是穩(wěn)定轉(zhuǎn)染（*轉(zhuǎn)染）。前者外源DNA/RNA不整合到宿主染色體中，因此一個(gè)宿主細(xì)胞中可存在多個(gè)拷貝數(shù)，產(chǎn)生高水平的表達(dá)，但通常只持續(xù)幾天，多用于啟動(dòng)子和其它調(diào)控元件的分析。一般來說，超螺旋質(zhì)粒DNA轉(zhuǎn)染效率較高，在轉(zhuǎn)染后24-72小時(shí)內(nèi)（依賴于各種不同的構(gòu)建）分析結(jié)果，常常用到一些報(bào)告系統(tǒng)如熒光蛋白，β半乳糖苷酶等來幫助檢測。

穩(wěn)定轉(zhuǎn)染，外源DNA既可以整合到宿主染色體中，也可能作為一種游離體（episome）存在。盡管線性DNA比超螺旋DNA轉(zhuǎn)入量低但整合率高。外源DNA整合到染色體中概率很小，大約1/104轉(zhuǎn)染細(xì)胞能整合，通常需要通過一些選擇性標(biāo)記，如來氨丙基轉(zhuǎn)移酶（APH；新霉素抗性基因），潮霉素B磷酸轉(zhuǎn)移酶（HPH），胸苷激酶（TK）等反復(fù)篩選，得到穩(wěn)定轉(zhuǎn)染的同源細(xì)胞系。轉(zhuǎn)染技術(shù)的選擇對(duì)轉(zhuǎn)染結(jié)果影響也很大，許多轉(zhuǎn)染方法需要優(yōu)化DNA與轉(zhuǎn)染試劑比例，細(xì)胞數(shù)量，培養(yǎng)及檢測時(shí)間等。

EZ Trans細(xì)胞轉(zhuǎn)染試劑是一種陽離子聚合物（PEI）細(xì)胞轉(zhuǎn)染液，是新一代的轉(zhuǎn)染試劑，帶正電的聚合物與核酸帶負(fù)電的磷酸基團(tuán)形成帶正電的復(fù)合物后與細(xì)胞表面帶負(fù)電的蛋白多糖相互作用，并通過胞吞作用進(jìn)入細(xì)胞。除了具有陽離子脂質(zhì)體（如：Lipo2000,3000）的轉(zhuǎn)染效率高，操作簡單，適用范圍廣，重復(fù)性好等特點(diǎn)外，還具有在體內(nèi)，轉(zhuǎn)染效率高，細(xì)胞毒性低等特點(diǎn)。

EZ Trans細(xì)胞轉(zhuǎn)染試劑一種具有較高的陽離子電荷密度的有機(jī)大分子，每相隔二個(gè)碳個(gè)原子都是質(zhì)子化的氨基氮原子，使得聚合物網(wǎng)絡(luò)在任何pH下都能充當(dāng)有效的“質(zhì)子海綿”體。其轉(zhuǎn)染性能好于樹枝狀聚合物，而且它的細(xì)胞毒性低.

EZ Trans細(xì)胞轉(zhuǎn)染試劑與其衍生物用作基因轉(zhuǎn)染載體的研究比分枝狀PEI（也寫作：BPEI, 24765-2）要早一些，過去的研究認(rèn)為在不考慮具體條件情況下，LPEI/DNA轉(zhuǎn)染復(fù)合物的細(xì)胞毒性較低，有利于細(xì)胞定位，轉(zhuǎn)染效率比BPEI高一些。但zui近研究表明BPEI的分枝度高有利于形成小的轉(zhuǎn)染復(fù)合物,從而提高轉(zhuǎn)染效率。本公司兩種PEI轉(zhuǎn)染液均可提供，您可根據(jù)文獻(xiàn)選擇使用。

幾種細(xì)胞轉(zhuǎn)染方法（試劑）特點(diǎn)比較表

運(yùn)輸與保存方法

常溫運(yùn)輸，4℃保存，保質(zhì)期12個(gè)月。

使用注意事項(xiàng):

質(zhì)粒質(zhì)量：請(qǐng)務(wù)必使用高質(zhì)量轉(zhuǎn)染級(jí)無內(nèi)毒素質(zhì)粒（推薦使用QIAGEN或者M(jìn)N公司去內(nèi)毒素質(zhì)粒提取試劑盒）。通過 260 nm 光吸收測定 DNA 濃度，260 nm / 280 nm 比值確定 DNA 純度（比值應(yīng)該在 1.8~2.0 的范圍之內(nèi)）。如有可能，請(qǐng)通過瓊脂糖凝膠電泳檢測質(zhì)粒的完整性。

細(xì)胞條件：使用適當(dāng)保存和經(jīng)常傳代的健康細(xì)胞。確保培養(yǎng)基沒有被細(xì)菌、真菌或支原體污染。如果細(xì)胞是近期復(fù)蘇的液氮凍存細(xì)胞，請(qǐng)?jiān)谵D(zhuǎn)染前至少傳代兩次。建議使用GIBCO或者78bio公司血清培養(yǎng)細(xì)胞。?

使用方法：

瞬時(shí)轉(zhuǎn)染方法

1. 接種細(xì)胞：轉(zhuǎn)染前一天，用膠原酶消化細(xì)胞并計(jì)數(shù)（不建議用胰酶）。調(diào)整細(xì)胞濃度，將細(xì)胞鋪入細(xì)胞培養(yǎng)的器皿，每個(gè)孔置入的細(xì)胞量應(yīng)能使轉(zhuǎn)染時(shí)細(xì)胞匯合度達(dá)到 70~80%。

2. 準(zhǔn)備 DNA-PEI 復(fù)合物： DNA、PEI 試劑和稀釋劑在進(jìn)行以下步驟前需先使其升至室溫。依據(jù)下表所示，用 Opti-MEM ITM（Invitrogen）或其他適合的無蛋白培養(yǎng)基稀釋適量 DNA。用同樣的培養(yǎng)基稀釋PEI 試劑。每 1 μg DNA 需用2-5 μL 線性PEI轉(zhuǎn)染試劑。一邊輕輕渦旋裝有 DNA 溶液的試管，一邊將稀釋的線性PEI轉(zhuǎn)染試劑滴加至試管中（注意：請(qǐng)勿顛倒添加順序）。充分混勻后，室溫靜置 10~25 min 以形成DNA-PEI復(fù)合物。當(dāng)溶液體積較大時(shí)，請(qǐng)用圓底聚丙烯管，例如 Falcon 5 mL /14 mL 離心管。

3. 轉(zhuǎn)染細(xì)胞：直接向每個(gè)孔中加入 DNA-PEI復(fù)合物并輕輕渦旋培養(yǎng)板/培養(yǎng)皿。在無血清條件下轉(zhuǎn)染時(shí)，去除生長培養(yǎng)基，替換成無血清培養(yǎng)基，然后滴DNA-PEI復(fù)合物。轉(zhuǎn)染 3 h 后，添加1/2體積的包含 30%血清的生長培養(yǎng)基。

4. 孵育細(xì)胞和分析結(jié)果：在 CO2培養(yǎng)箱中 37℃下孵育細(xì)胞至可以分析檢測。轉(zhuǎn)染后zui快 7h 即可檢測到轉(zhuǎn)入基因的表達(dá)。請(qǐng)自行確定檢測時(shí)間。?

穩(wěn)定轉(zhuǎn)染方法

1. 接種細(xì)胞：轉(zhuǎn)染前一天，用胰酶消化細(xì)胞并計(jì)數(shù)。調(diào)整細(xì)胞濃度，將細(xì)胞鋪入細(xì)胞培養(yǎng)的器皿，總體積如表 1 所示，每個(gè)孔置入的細(xì)胞量應(yīng)能使轉(zhuǎn)染時(shí)細(xì)胞匯合度達(dá)到 70~80%。

2. 準(zhǔn)備 DNA-PEI 復(fù)合物： DNA、PEI 試劑和稀釋劑在進(jìn)行以下步驟前需先使其升至室溫。依據(jù)表 1 所示，用 Opti-MEM ITM（Invitrogen）或其他適合的無蛋白培養(yǎng)基稀釋適量 DNA。用同樣的培養(yǎng)基稀釋PEI 試劑。每 1 μg DNA 需用2-5 μL 線性PEI轉(zhuǎn)染試劑。一邊輕輕渦旋裝有 DNA 溶液的試管，一邊將稀釋的線性PEI轉(zhuǎn)染試劑滴加至試管中（注意：請(qǐng)勿顛倒添加順序）。充分混勻后，室溫靜置 10~25 min 以形成 DNA-PEI復(fù)合物。當(dāng)溶液體積較大時(shí)，請(qǐng)用圓底聚丙烯管，例如 Falcon 5 mL /14 mL 離心管。

3. 轉(zhuǎn)染細(xì)胞：直接向每個(gè)孔中加入 DNA-PEI復(fù)合物并輕輕渦旋培養(yǎng)板/培養(yǎng)皿。在無血清條件下轉(zhuǎn)染時(shí)，去除生長培養(yǎng)基，替換成無血清培養(yǎng)基，然后滴DNA-PEI復(fù)合物。轉(zhuǎn)染 3 h 后，添加½體積的包含 30%血清的生長培養(yǎng)基。

4. 孵育細(xì)胞和分析結(jié)果：在 CO2培養(yǎng)箱中 37℃下孵育細(xì)胞至可以分析檢測。轉(zhuǎn)染后zui快 7h 即可檢測到轉(zhuǎn)入基因的表達(dá)。

5.轉(zhuǎn)染 24 h 后，將細(xì)胞傳代至新鮮的生長培養(yǎng)基中（將細(xì)胞稀釋 10 倍以上），在 CO2培養(yǎng)箱中 37℃孵育12小時(shí)。第二天加入與轉(zhuǎn)染抗性基因相匹配的篩選藥物。約 1~2 周可篩選到耐藥性克隆，在這期間需經(jīng)常更換含篩選藥物的生長培養(yǎng)基。?

特別提醒:

1. 對(duì)于某些類型的細(xì)胞如 HEK-293、HEK293T、NIH/3T3 和 COS 細(xì)胞，在轉(zhuǎn)染前兩天鋪板可顯著提高轉(zhuǎn)入基因的表達(dá)水平。如果選擇轉(zhuǎn)染前兩天鋪板，可適當(dāng)降低鋪板密度，以確保轉(zhuǎn)染時(shí)細(xì)胞的匯合度仍為 70~80%。

2. 對(duì)于接觸抑制敏感的細(xì)胞，可適當(dāng)降低鋪板密度。

3. 即使在有蛋白（如 10%的血清）存在的情況下，DNA-PEI復(fù)合物仍能轉(zhuǎn)染細(xì)胞，但是 DNA-PEI復(fù)合物必須在無蛋白存在的條件下形成。我們推薦使用 Opti-MEM ITM 培養(yǎng)基以達(dá)到*轉(zhuǎn)染效率。其他的無蛋白培養(yǎng)基則需測試與線性PEI轉(zhuǎn)染試劑的兼容性。

4. 對(duì)大多數(shù)細(xì)胞來而言，每 1 μg DNA 使用 3.0 μLEndoFectinTM 試劑都能獲得較高轉(zhuǎn)染效率。使用者也可嘗試每 1 μg DNA 使用 1~4 μL 體積線性PEI轉(zhuǎn)染試劑進(jìn)行優(yōu)化。

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