Recombinant DNA Technology

Recombinant DNA

Technology

Dr. Hui LIOffice : S408Tel: 26538722

Topic 3

Transfer of target gene into receptor cell

Bacteria that are capable of uptaking the foreign DNA are called competent;

To increase the permeability of cell membrane.

How to make competent E.coli cells?

Procaryote cells as receptor cells

Use restriction enzymes-deficiency E.coli

Species

Calcium chloride (CaCl2)transformation is a method of promoting competence for bacterial cells.

Principle

Procedure

E.coli culture

Until OD600 0.3-0.4

On ice 5-10 min

Collection of bacterial cells by centrifugation at 4 ℃

Suspension of cell pellet with chill CaCl2

Collect cells by centrifugation at 4 ℃

Store at -70 ℃

On ice 30 min

Suspension of cell pellet with chill CaCl2

Collect cells by centrifugation at 4 ℃

Suspension of cell pellet with chill CaCl2

How to introduce foreign DNA?

（ 1 ）转化（ transformation ）

（ 2 ）转染（ transfection)

大肠杆菌捕获噬菌体 DNA 的过程。

Methods of introduction foreign DNA into competent cells

（ 3 ）转导（ transduction)

借助噬菌体把外源 DNA 导入细菌的过程。

大肠杆菌捕获质粒 DNA 的过程。

Number of colonies / g DNAAn excellent preparation of competent cells will give ~108 colonies per microgram of plasmid.

Methods of transformation

Efficiency

简单，但转化效率不高（ 106-108/g DNA ）。

（ 1 ） Heat shock( 热休克法）

转化效率高（高于 109/g DNA ）。

（ 2 ） Electroporation( 电转化法 )

10ng vector

100L competent cells

On ice, 10 min

42ºC 30s-120s

Add 1mL LB cultureIncubate at

37ºC for 1h10-100 L plated on antibiotic selective media (Only transformants can survive)

Adhesion of DNA Uptake of DNA

（ 1 ） Heat shock

Electroporation is another method of promoting competence. In the method the cells are briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell membrane through which the plasmid DNA enters. This method is amenable to the uptake of large plasmid DNA. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.

（ 2 ） Electroporation

Bacterial culture on

plate

Circular recombinant plasmid

Self-ligation of plasmid

* Linear plasmids will be degraded once they enter into bacteria

① circular plasmid

（ 1 ） Recombinant vector

Factors affected efficiency of recombination

①Use the cells at exponential phase

②Prepare under ice condition

④Store at -70℃

③Treat sufficiently with CaCl2

⑤Thaw the cells quickly, and never re-store the cells after thawing

（ 2 ） competent cells

把重组的噬菌体 DNA 或 Cosmid 质粒DNA 包装成具有感染能力的噬菌体颗粒。

Transduction by phages

（ 1 ） in vitro packaging （体外包装）

（ 2 ） Transduction （转导）

通过受体菌细胞表面的 DNA 接受器位点（ receptor site) ，使带有外源基因的重组体 DNA 注入受体大肠杆菌进行扩增。

cI857 基因是个温度敏感性阻遏蛋白基因，感染细菌后，在 32℃ 下培养细菌时能够保持溶源性。

但当温度升高到 44 —45℃ ℃ 时，就会导致 cI 基因编码的阻遏蛋白失活， DNA复制、外壳蛋白合成。便于提取外壳蛋白。

（ 3 ） cI857 基因突变的噬菌体

但由于该噬菌体的 S 基因上有一个突变，所以细菌还不裂解。

cI857 其它基因

溶源状态（ DNA 不转录、不翻译）

DNA

阻遏蛋白

阻遏蛋白

44 —45℃ ℃

DNA 转录、翻译合成外壳蛋白

32℃

噬菌体 1 外壳蛋白基因 E 发生了无义突变，不能合成头部蛋白，但能合成其它外壳蛋白（尾部蛋白）。

在 cI857 基因突变的噬菌体的基础上，选择两种外壳蛋白的突变型噬菌体。

（ 4 ） 互补型噬菌体

外壳蛋白基因 D 发生了无义突变，不能合成头部的包装识别蛋白，但能合成其它外壳蛋白（头部、尾部蛋白）。

噬菌体 2

(5) 体外包装过程

体外包装好的重组噬菌体感染受体菌，使受体菌发生溶菌，形成噬菌斑。（每 g DNA 能形成 106 噬菌斑）。

（ 6 ） 转导

Eukaryote cells as receptor cells

I. Yeast cells

II. Plant cells

III. Animal cells

（ 1 ） Protoplast methode

Methods of transformation for yeast cells

yeast protoplast competemt

Degrade Cell wall(chitinase)

CaCl2 、PEG

Vector containing foreign DNA

PEG（聚乙二醇）使细胞壁具有通透性，允许 DNA进入。CaCl2使细胞膜具有通透性，允许 DNA进入。

transformation

I. Yeast cells

yeast0.1mol/L LiCl

(Lithium chloride )

Vector containing foreign DNA

competent

40%PEG 4000

（ 2 ） Li+ method

叶盘

消毒 切取 土壤农杆菌浸泡

看护培养基

愈伤组织

分化幼苗

II. Plant cells

1. leaf disk mediated by Agrobacterium ( 叶盘法 )

Plant cells protoplastCellulase or pectinase( 纤维素酶和果胶酶 )

DNA

Mix with buffer of electroporation

1-2kV,3-25F

愈伤组织(Callus)

幼苗(shoots)

2. Electroporation ( 电击法 )

make transient holes in cell membranes using electric shock

Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. The transformation efficiency is lower than in agribacterial mediated transformation, but most plants can be transformed with this method.

3. Gene gun （基因枪法）

（ 1)Principle ：

III. Animal cells

1. Calcium phosphate precipitation

HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.

（ 2 ） No need to use vectors

2. Liposome （脂质体）

small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. For eukaryotic cells, transfection is better achieved using cationic liposomes (or mixtures), because the cells are more sensitive.

脂质体有商业试剂盒（如 Life Technologies）

Foreign DNA is directly injected into the nucleus of receptor cells

3. Microinjection ( 显微注射法 )

二乙氨乙基（ diethyl-aminoehtyl ， DEAE ）葡萄糖能促进哺乳动物细胞摄入外源 DNA 。The negatively charged DNA binds to DEAE and the complex is taken up by the cell via endocytosis.

4. DEAE-dextran (DEAE--- 葡萄糖传染法 )

吸附到细胞表面后被细胞吞入，部分 DNA可以进入到细胞核里。

DEAE-dextran 细胞 外源 DNA预处理 摄入

DEAE-dextran

外源 DNA细胞混合

DEAE-dextran is toxic to cells!

5. Via virus

18