Gene cloning, expression

and functional study

基因克隆，表达及功能研究

vectorsvectors

Cloning vectors: 克隆载体to clone a gene in a vector

Expression vectors: 表达载体to express a gene from a vector

Integration vectors: 整合载体to integrate a gene in a genome thr

ough a vector

Cloning vectors: 克隆载体to clone a gene in a vector

Expression vectors: 表达载体to express a gene from a vector

Integration vectors: 整合载体to integrate a gene in a genome thr

ough a vector

Cloning vectors Cloning vectors

1 Plasmid vecters

2 Bacteriophage vectors

3 Cosmids & BACs

4 Eukaryotic vectors

Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.

expression vectors: allowing the exogenous DNA to be inserted, stored, and expressed.

1. Contains an origin of replication, allowing for replication independent of host’s genome.

2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistanceblue-white screening

3. Contains a multiple cloning site (MCS)4. Easy to be isolated from the host cell.

A plasmid vector for cloning A plasmid vector for cloning

Ampicillin resistant? yes yesTetracycline resistant? No yes

B X B

B

B

XAmpr

ori

Ampr

Tcr

ori

pBR322

Ampr Tcr

ori

-Screening by insertional inactivation of a resistance geneTwin antibiotic resistance screeningTwin antibiotic resistance screening

Replica plating: transfer of the colonies from one plate to another using absorbent pad or Velvet ( 绒布 ).

transfer of colonies

+ampicillin + ampicillin+ tetracycline

these colonies have bacteria with recombinant plasmid

Blue white screeningBlue white screening

Ampr

ori

pUC18(3 kb)

MCS (Multiple cloning sites,多克隆位点）

Lac promoter

lacZ’

Screening by insertional inactivation of the lacZ gene

The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal.

Recreated vector: blue transformantsRecombinant plasmid containing inserted DNA: white transformants

Recreated vector (no insert)

Recombinant plasmid (contain insert)

back

Multiple cloning sitesMultiple cloning sitesMultiple restriction sites enable the convenient insertion of target DNA into a vector

Ampr

ori

pUC18(3 kb)

MCS (Multiple cloning sites,多克隆位点）

Lac promoter

lacZ’

…ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA…

. T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser…

EcoRI SacI KpnISmaIXmaI BamHI XbaI

SalIHincIIAccI PstI SphI

Lac Z

A plasmid vector for gene expressionA plasmid vector for gene expression

Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed.

1. Promoter and terminator for RNA transcription are required.

2. Intact ORF and ribosomal binding sites (RBS) are required for translation.

3. Include ： (1) bacterial expression vectors, (2) yeast expression vectors, (3) mammalian expression vector

T7 promoter

RBSStart codon

MCS

Transcription terminator

Ampr

ori

T7 expression

vector

An bacterial expression vectorAn bacterial expression vector

MCS

A yeast expression vectorA yeast expression vector

Bacteriophage vector Bacteriophage vector

Two examples:1.λ phage • bacteriophageλ• λ replacement vector 2.M13 phage• M13 phage vector• Cloning in M13• Hybrid plasmid-M13 vectors

• viruses that can infect bacteria. •48.5 kb in length•Linear or circular genome (cos ends)•Lytic phase (Replicate and release)•Lysogenic phase (integrate into host genome)

λ phageλ phage

Analysis of eukaryotic genes and the genome organization of eukaryotes requires vectors with a larger capacity for cloned DNA than plasmids or phage .

Human genome (3 x 109 bp): large genome and large gene demand vectors with a large size capacity.

Cloning large DNA fragmentsCloning large DNA fragments(Eukaryotic Genome project)

Genomic library VS cDNA libraryGenomic library VS cDNA library

Cosmid vectorsCosmid vectors

1. Utilizing the properties of the phage cos sites in a plasmid vector.

2. A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the head.

3. The insert can be 37-52 kb

Digestion

Ligation

C) Packaging and infect

Formation of a cosmid clone

YAC vectorsYAC vectors

Accommodates genomic DNA fragments of more than 1 Mb, and can be used to clone the entire human genome, but not good in mapping and analysis.

(yeast artificial chromosome)

Essential components of YAC vectors :• Centromers (CEN), telomeres (TEL) and autonomous replicating sequence (ARS) for proliferation in the host cell. • ampr for selective amplification and markers such as TRP1 and URA3 for identifying cells containing the YAC vector in yeast cells. • Recognition sites of restriction enzymes (e.g., EcoRI and BamHI)

YAC Cloning

BAC vectors 细菌人工染色体

1. More stable than YAC2. Capacity is 300-350 kb3. One to two copies in each cell4. Easy to handle5. More popular in genomic mapping

I1 Genomic libraries

I1-1 Representative gene libraries

I1-2 Size of library I1-3 Genomic DNA I1-4 Vectors

Gene libraries and screening

Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis.

Genomic libraries

cDNA libraries

Gene library (made from genomic DNA)

(made from cDNA- copy of mRNA)

I1 Genomic

libraries

I1-1 Representative gene libraries

--- Contain all the original sequences

1. Certain sequences have not been cloned.Example: repetitive sequences lacking restriction sites

2. Library does not contain sufficient clones

Missing original sequence

Too long for the vector used

I1 Genomic

libraries

I1-2 Size of library (ensure enough clones)

must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence The formula to calculate the number of recombinants:

N = ln (1-P)

ln (1-f)

P: desired probability f : the fraction of the genome in one insert

I1 Genomic

libraries

For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are :

N E.coli= = 1.1 ×103 ln( 1-0.99)

ln[1-(2×104/4.6×106)]

Nhuman= = 6.9 ×105 ln(1-0.99)

ln[1-(2 ×104/3 ×109)]These values explain why it is possible to make good genomic libraries from prokaryotes in plasmids where the insert size is 5-10kb ,as only a few thousand recombinants will be needed.

I1 Genomic

libraries

I1-3 Genomic DNA libraries

Purify genomic DNA

Fragment this DNA : physical

shearing and restriction enzyme

digestion

eukaryotes

prokaryotes

Clone the fragments into vectors

I1 Genomic

libraries

To make a representative genomic libraries ,genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning into the chosen vector.

Purification of genomic DNA :

Prokaryotes :extracted DNA directly from cells

remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction.

Eukaryotes :prepare cell nuclei

I1 Genomic

libraries

Break DNA into fragments randomly:

Physical shearing : pipeting, mixing or sonicaion

Restriction enzyme digestion: partial digestion is

preferred to get a greater lengths of DNA fragments.

I1 Genomic

libraries

Sau3A: 5’-/GATC-3’, less selectivityBamH1: 5’-G/GATCC

Selection of restriction enzyme1. Ends produced (sticky or blunt) &

The cleaved ends of the vector to be cloned

2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals

3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.

I1 Genomic

libraries

I1-4 Vectors

According to genome’s size,we can select a proper vector to construct a library .

Vectors Plasmid phageλ cosmid YAC insert (kb) 5 23 45 1000

The most commonly chosen genomic cloning vectors are λ relacement vectors which must be digested with restriction enzymes to produce the two λ end fragment or λ arms between which the genomic DNA will be digested

I1 Genomic

libraries

cos cos

Long (left)arm

short (right)arm

Exogenous DNA(~20-23 kb)

λ phage vector in cloning

cos cos

Long (left)arm

short (right)arm

Exogenous DNA(~20-23 kb)

λ replacement vector cloning

2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes 3. Infection and

formation of plaques

Library constructed

1. Ligation

0.preparation of arm and genomic inserts

I 2 cDNA librariesI2-1 mRNA isolation, purification

I2-2 Check theRNA integrity

I2-3 Fractionate and enrich mRNA

I2-4 Synthesis of cDNA

I2-5 Treatment of cDNA ends

I2-6 Ligation to vector

Gene libraries and screening

cDNA libraries

1.No cDNA library was made from prokaryotic mRNA.

• Prokaryotic mRNA is very unstable• Genomic libraries of prokaryotes ar

e easier to make and contain all the genome sequences.

I 2 cDNA libraries

2.cDNA libraries are very useful for eukaryotic gene analysis

• Condensed protein encoded gene libraries, have much less junk sequences.

• cDNAs have no introns genes can be expressed in E. coli directly

• Are very useful to identify new genes• Tissue or cell type specific (differential ex

pression of genes)

cDNA libraries

I 2 cDNA libraries

I2-1 mRNA isolation

• Most eukaryotic mRNAs are polyadenylated at their 3’ ends

• oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.

AAAAAAAAAAn5’ cap

I 2 cDNA libraries

I 2 cDNA libraries

1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose

2.More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet 3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients

Three methods to isolate mRNA.

I2 cDNA libraries

Make sure that the mRNA is not degraded. Methods:Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translatedAnalysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels

I2-2 Check the mRNA integrity

I2 cDNA libraries

I2-3 Cloning the particular mRNAs

Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library.

Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gelsEnrichment: carried out by hybridizationExample: clone the hormone induced mRNAs (substrated cDNA library)

I2 cDNA libraries

I2-4 Synthesis of cDNA :

First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1)

Second strand synthesis: best way of making full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primer

to make the second. (Fig2.1)

I2 cDNA libraries

5’ mRNA AAAAA-3’ HO-TTTTTP-5’

5’

Reverse transcriptaseFour dNTPs

AAAAA-3’TTTTTP-5’

mRNA

mRNA

cDNA

cDNA

cDNA

Duplex cDNA

AAAAA-3’

TTTTTP-5’

TTTTTP-5’

3’

3’-CCCCCCC

Terminal transferasedCTP

Alkali (hydrolyaes RNA)Purify DNA oligo(dG)

Klenow polymerase or reverseTranscriotase Four dNTPs

5’-pGGGG-OH

5’

3’-CCCCCCC

5’-pGGGG3’-CCCCCCC TTTTTP-5’

-3’

Fig 1.1 The first strand synthesis

I2 cDNA libraries

5’-pGGGG3’-CCCCCCC

HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC

5’-pAATTCGGGGGG

TTTTTGGCTTAAGCC-OH CCGAATTCGG-3’

3’-CCCC

3’-CCCCCCC

3’-CCC 5’-pGGGG

5’-pGGGG

TTTTTp-5’ -3’

TTTTTp-5’

TTTTTp-5’

-3’

-3’

TTTTTGGCTTAAp-5’

HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH

CCG-3’

Duplex cDNA

Single strand-specific nuclease

Klenow polymerase

treat with E.coRI methylase

Add E.colRI linkers using T4 DNA ligase

E.colRI digestion

Ligate to vector and transfom

Fig2.1 Second strand synthesis

I2-5 Treatment of cDNA endsBlunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.

The process :

Move protruding 3’-ends(strand-special nuclease)

Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs)

Ligate the blunt-end and linkers(T4 DNA ligase)

Restriction enzyme digestion (E.coRI )

Tailing with terminal transferase or using adaptor molecules

I2 cDNA libraries

I2-6 Ligation to vector Any vectors with an E.coRI site would suitablefor cloning the cDNA.

The process :

Dephosphorylate the vector with alkalinephosphatase

Ligate vector and cDNA with T4 DNA ligase

(plasmid or λ phage vector)

I2 cDNA libraries

I3 Screening proceduresI3-1 Screening

I3-2 Colony and plaque hybridization

I3-3 Expression screening

I3-4 Hybrid arrest and release

I3-5 Chromosome walking (repeat screening)

Gene libraries and screening

I3-1 Screening

The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .

1. Using nucleic acid probe to screen the library based on hybridization with nucleic acids.

2. Analyze the protein product.

I3 Screening procedures

Screening libraries

Hybridization to identify the interested DNA or its RNA product

1. Radiolabeled probes which is complementary to a region of the interested geneProbes: • An oligonucleotide derived from the

sequence of a protein product of the gene• A DNA fragment/oligo from a related gene

of another species 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA

membrane (Southern) or RNA (Northern) membrane

Searching the genes of interest in a DNA library

I3 Screening procedures

I3-2 Colony and plaque hybridization

Transfer the DNA in the plaque or colony to aNylon or nitrocellulose membrane

Phage DNA bind to the membrane directly

Bacterial colonies must be lysed to release DNA on the membrane surface.

Hybridization (in a solution Containing Nucleic acid probe)

Wash to remove unhybri-dization probe and visualize

X-ray film(radio-actively labeled )

antibody or enzyme(modified nucleotide labeled

Line up the hybridizated region orrepeated hybridization

(Alkali treatment)

I3 Screening procedures

Transfer to nitrocelluloseor nylon membrane

Denature DNA(NaOH)Bake onto membrane

Probe with 32p-labled DNA complementary to gene of interest

Expose to film

Select positive from master plate

Keep master plate

Screening by plaque hybridization

I3 Screening procedures

Identify the protein product of an interested gene

1.Protein activity2.Western blotting using a

specific antibody

I3 Screening procedures

I3-3 Expression screening

Expression screening (1)

If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. However, this screening may miss the right clone

Example: the EcoRI site of lgt11 vector. The inserted genes have one in six change (1/6) to be in both the correct orientation (2 possibilities; ) and reading frame (three possibilities; three nucleotide code XXX).

I3 Screening procedures

Expression screening (2)

The procedure has similarities to the plaque hybridization protocol.

‘Plaque lift’ ( taken by placing a membrane on the dish of plaque)

Immersed in a solution of the antibody

Detected by other antibodies

Repeat cycles of screening to isolate pure plaques

Antibodies can be used to screen the expression library.

I3 Screening procedures

基因表达

1. Prokaryotic expression vector

原核表达载体2. Baculovirus expression vector

昆虫杆状病毒表达载体3. Mammalian expression vector

哺乳动物表达载体4. Adenoviral and retroviral vector

腺病毒及逆转录病毒表达载体

Prokaryotic expression vector

原核表达载体

GST-fusion

6xHis-fusion

GST

HIS

基因功能研究

1. Overexpression in cells 超表达，观察表型2. RNAi 干扰技术3. Yeast two hybrid system 酵母双杂交等技术

寻找与目的基因相关的蛋白4. Protein expression and antibody preparation

表达蛋白与抗体制备5. Localization of protein 蛋白在细胞中的定位

The end