基因轉(zhuǎn)染實(shí)驗(yàn)相信實(shí)驗(yàn)室的大多數(shù)小伙伴都有做過(guò)，目前, 將外源DNA導(dǎo)入哺乳動(dòng)物細(xì)胞的方法大致可分為兩大類，即生物方法和物理化學(xué)方法。生物方法主要是以病毒作為載體,通過(guò)病毒感染的方式將外源DNA 導(dǎo)入到細(xì)胞中，物理化學(xué)方法常用的有磷酸鈣法、顯微注射、電穿孔、脂質(zhì)體，以及高分子聚合物為載體的基因轉(zhuǎn)染技術(shù)。

本文介紹的是一種陽(yáng)離子高分子聚合物-------聚乙烯亞胺（Polyethyleneimine，PEI），其是一種具有較高陽(yáng)離子電荷密度的有機(jī)大分子，它每第三個(gè)原子都是質(zhì)子化的氨基氮原子，使得該聚合物在任何pH下都能充當(dāng)有效的質(zhì)子海綿體。它能將DNA縮合成帶正電荷的微粒，這些微粒可以黏合到帶有負(fù)電荷的細(xì)胞表面殘基，并通過(guò)胞吞作用進(jìn)入細(xì)胞。聚合物進(jìn)入細(xì)胞后，胺的質(zhì)子化導(dǎo)致反離子大量涌入以及滲透勢(shì)降低，上述變化導(dǎo)致的滲透膨脹使囊泡釋放聚合物與DNA形成的復(fù)合物進(jìn)入細(xì)胞質(zhì)，復(fù)合物拆解后，DNA就能自由的融合到細(xì)胞核中。PEI是歷*繼多聚賴氨酸之后發(fā)現(xiàn)的第二種聚合物轉(zhuǎn)染試劑。聚乙烯亞胺有線性和支鏈兩種，有多種分子量可選，實(shí)驗(yàn)證明線性和支鏈都具有轉(zhuǎn)染效果，而分MW為25000的線性PEI具有較高的轉(zhuǎn)染效率。后期科研人員又對(duì)該分子進(jìn)行了優(yōu)化，對(duì)其分子進(jìn)行了修飾，制出了MW為40000的線性PEI，使其溶解性顯著提高，轉(zhuǎn)染效率更高，細(xì)胞毒性更低。線性PEI廣泛適用于常見(jiàn)細(xì)胞系，如：HEK-293、HEK293T、Hep G2、Hela、CHO-K1、COS-1、COS-7、HIH/3T3和sf9等，PEI即使在有血清存在的情況下仍然能夠的將核酸導(dǎo)入細(xì)胞，其具有*的轉(zhuǎn)染效率，重組蛋白的高表達(dá)水平，且與含血清的培養(yǎng)基相兼容，低細(xì)胞毒性，PEI的另外一個(gè)顯著優(yōu)勢(shì)是較其他轉(zhuǎn)染試劑使用成本極低。

產(chǎn)品特性

CAS號(hào)：9002-98-6

分子式：(C2H5N)x

熔點(diǎn)：73-75℃ 

溶解性：溶于熱水、低pH冷水, 甲醇和乙醇。不溶于乙mi、丙酮和冷水。

PEI MW25000操作說(shuō)明：

Transfection Reagent Preparation and Storage Recommendations，These guidelines are our recommendations based off research, testing, and customer feedback. There are alternative methods available, but we suggest following these recommendations for easy adoption and consistently high performance. Thesemethods are adapted from Cold Spring Harbor Protocols, an excellent secondary resource for transient transfection literature.

Reagent Preparation (1 mg/mL)

Materials:

1.1g PEI 25K (kingmorn Catalog# KE1075).
2.1L Milli-Q® water, water for injection (WFI), or comparable biological-grade water
3.12 M Hydrochloric Acid (HCl)
4.10 M Sodium Hydroxide (NaOH)
5.Disposable 0.1-0.2um PES vacuum sterile-filter.

6.Sterile HDPE or polypropylene storage vials for reagent aliquots. 

Equipment：

1.1L glass beaker
2.1L glass graduated cylinder
3.Calibrated pH meter
4.2 x disposable 1mL plastic pipettes
5.Stir plate
6.PTFE coated stir bar
7.Vacuum pump
Method:
1.Dispense 1g of PEI 25K into beaker and suspend in 900 mL of water.
2.Add stir bar and set stirring to produce a small vortex.
3.Add hydrochloric acid dropwise until pH is< 2.0.
4.Cover top of beaker and stir for up to 3 hours until powder fully dissolves.
5.Add sodium hydroxide dropwise until pH is 6.9-7.1.
6.Transfer solution to graduated cylinder and add water until the total volume is 1L.
7.Sterile-filter through vacuum membrane.

8.Aliquot as desired and store at -20℃.

Reagent Storage：
Frozen aliquots can be stored at -20°C for up to one year. Aliquots can be thawed and kept at 4°C for up to two weeks, but should never be re-frozen.
References
1. Polyethylenimine (PEI), linear (1 mg/mL). Cold Spring Harbor Protocols 2008, pdb.rec11323– pdb.rec11323 (2008). doi:10.1101/pdb.rec11323

PEI MW4000操作說(shuō)明：

Transfection Reagent Preparation (1 mg/mL)

Materials：

1.1g PEI MAX 40K (kingmorn Catalog#KE1098)

2.1L Milli-Q® water, water for injection (WFI), or comparable biological-grade water

3.1N Sodium Hydroxide, USP

4.Sterile 25 mL plastic pipette

5.0.1µm, 0.2µm, or 0.22µm PES vacuum sterile-filter unit

6.Sterile HDPE or polypropylene storage vials

Equipment：

1.1L glass beaker

2.PTFE-coated stir bar

3.Magnetic stir plate

4.1L glass graduated cylinder

5.pH meter

6.Pipette controller

7.Vacuum pump with tubing

Method：

1.In 1L glass beaker, suspend 1g of PEI MAX 40K in 900 mL water.

2.Add PTFE-coated stir bar to 1L glass beaker and set stirring to produce small vortex.

3.Wait for PEI MAX 40K to completely dissolve. This typically takes less than 5 minutes.

4.Use 25 mL plastic pipette to add 1N sodium hydroxide dropwise to 1L glass beaker until pH is 6.90 to 7.10.

5.If pH accidently exceeds 7.10, use 1N hydrochloric acid and a new 25 mL plastic pipette to lower pH back to 6.90 to 7.10.

6.Decant solution from 1L glass beaker to 1L graduated cylinder.

7.Add water to 1L graduated cylinder to adjust final volume to 1L.

8.Sterile-filter solution through vacuum membrane.

9.Aliquot solution as desired.

10.Store aliquots at 4°C

Transfection Solution Storage and Shelf Life

At 4°C in a suitable container, the transfection solution will retain its maximum performance for at least six months. For qualitative work, the solution may be suitable for use for up to a year.