Part I: Gene Transfer Transduction,Transfection and Transformation Transduction-transfer of...

Part I: Gene Transfer Transduction ,Transfection and Transformati

on• Transduction-transfer of bacterial gene from one ba

cterium to another by a bacteriophage.

• Transfection-the uptake of DNA by a eukaryotic cell, followed by the incorporation of genetic markers present in the DNA into the cell’s genome.

• Transformation-genetic alteration of an organism brought about by the incorporation of foreign DNA in to cells.

Streptococcus pneumoniae stains

Streptococcus pneumoniae

Transformation in

Streptococcus

pneumoniae

CaCl2 induced transformation

Phage DNA and Packaging

Microinjection of a Protoplast-derived Potato Cells

Photograph of Microinjection

Biolistic Apparatus—Gene Gun

Structure of Gene Gun

Part II: Selection, screening and analysis of recombinants

• Selection: some sort of pressure (e.g. the pressure of an antibiotic) is applied during the growth of host cells containing recombinant DNA.

• Screening is a procedure by which a population of viable cells is subjected to some sort of analysis that enable the desired sequences to be identified.

Insertion inactivation selection in plasmid pBR322

Insertion inactivation

Antibiotic resistance and insertion inactivation

Iodine analysis of ampicillin resistant insertional inactivation

Clone and selection of lacY gene

Formation of R-loop structure

Antibiotics resistance selection and genetic complementation

Insertion inactivation

selection in plasmid

pUC18

Insertion inactivation in the αcomplementation system

Structure of X-gal

Different colonies in the use of X-gal

Insertion inactivation in vector λgt10 and Charon 16A

Insertion inactivation in vector λgt10

Charon Recombinant Packaging and Selection

Spi Selection system for Bacteriophage vector EMBL4

Colonies of E. coli

Bacteriophage plaques

Map of the yeast artificial chromosome vector pYAC2

Cloning in a YAC Vector

CPSF 基因在真核表达载体上的克隆

研究表明 , 至少 6 种蛋白复合体参与前体 mRNA3’ 端加工• 即切割与多聚腺嘌呤化特异性因子 (cleavage and polyadenylation s

pecificity factor,CPSF)• poly(A) 多聚酶 (poly(A) polymerase,PAP)• 切割刺激因子（ cleavage stimulation factor,CstF ）• 切割因子 I 和 II (cleavage factor I and II, CF Im and CF IIm) • 核内多聚腺苷酸结合蛋白 1 （ polyadenylate binding-protein nuclear

1 ,PABPN1 ）。 其中， CPSF 被认为在前体 mRNA3’ 端加工过程中起着核心作用，

直接与切割 / 多聚腺苷酸化信号 AAUAAA 结合，且与此序列一样，它在两个亚反应中都是必需的。

切割和多聚腺苷酸化信号示意图

C 末端与 N 末端 TAP-tag

Tandem Affinity Purification ， TAP

TAP 技术纯化蛋白质复合体流程示意图

切割复合体模型

多聚腺嘌呤化复合体模型

载体 pTRE2hyg 的形体结构和多克隆位点

载体 pTRE2hyg 的形体结构和多克隆位点图

载体 pTRE2hyg-TAP-tag 的形体结构和多克隆位点图

载体 pTRE2hyg-TAP-tag-CPSF 30K 的形体结构和多克隆位点

载体 pTRE2hyg-TAP-tag-CPSF 73K 的形体结构和多克隆位点

载体 pTRE2hyg-TAP-tag-CPSF 100K 的形体结构和多克隆位点

载体 pTRE2hyg-TAP-tag-CPSF 160K

的形体结构和多克隆位点图

质粒 pBS1761 的形体结构图

质粒 pUK 的形体结构图

TAP-tag 片段的 PCR 扩增

• Primer1 ： 5’ CGG GAT CCA TGG BamHI 切点

CAG GCC TTG CGC AAC 3’

• Primer2 ： 5’ GTC GAC GGC TAG NheI 切点

CTT ATC GTC ATC ATC AAG TGC3’

CPSF 基因的 PCR 扩增

• CPSF 30k Primer1 ： 5’GCT AGC CAT GCA GGA AAT CAT CGC C3’ NheI 切点• CPSF 73k Primer1 ： 5’GCT AGC CAT GTC CGC GAT TCC CGC3’ NheI 切点• CPSF 100k Primer1 ：5’GCT AGC CAT GAC ATC TAT TAT CAA ATT AAC TA3’ • NheI 切点• CPSF 160k Primer1 ：5’GCT AGC CAT GTA CGC TGT GTA CAA GCA3’ NheI 切点

四种 CPSF 基因通用 Primer2 ：

• 5’AAG CTT GCG GCC GCT TCG GGC TTT GTT AGC AGC3’

• 四种 CPSF 基因的 Primer1 以其对应的基因 5’ 端序列为基准进行设计的，而通用Primer2 是以 CPSF 基因下游的 pUK 载体序列为基准进行设计的。

Eukaryotic Expression Vector and Restriction Enzyme Digestion

Screening clone banks by nucleic acid hybridisation

Screening plaques at high and low densities

Immunological Screening for Expressed Genes

Hybrid arrest and hybrid release translation to identify the protein product of a cloned

fragment

Blotting apparatus

Southern Blotting

Nobel Prize 2006

Andrew Fire Craig Mello

RNAi (RNA interference)- 一些小的双链 RNA 可以高效、特异的阻断体内特定基因表达，促 使 mRNA 降解，诱使

细胞表现出特定基因缺失的表型，称为 RNA 干扰

诺贝尔奖评审委员会评语： 他们的发现能够解释许多令人困惑、相互矛

盾的实验观察结果，并揭示了控制遗传信息流动的自然机制。这开启了一个全新的研究领域，未来这种技术能用来直接从源头上让致病基因“沉默”，更有效的治疗癌症甚至艾滋病，在农业上也将大有可为。

Gene Transfer for Chalone Synthetase (cosuppression),1990

1995, Guo’s experiment Antisense RNA

Caenorhabditis elegans

par-1 gene sense RNA,

blocking gene expression

1998, Andrew Fire and Craig Mello

• Organism: Caenorhabditis elegans

• RNA: dsRNA

• Result: degradation of mRNA

• Process:

RISC: RNA-induced

silencing complex

RNA 沉默机制

• RNA 沉默机制