Gene cloning, expression and functional study 基因克隆，表达及功能研究

vectors 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 through a vector

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.

A plasmid vector for cloning Contains an origin of replication, allowing for replication independent of host’s genome. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening Contains a multiple cloning site (MCS) Easy to be isolated from the host cell.

Twin antibiotic resistance screening -Screening by insertional inactivation of a resistance gene Ampr Tcr ori pBR322 B X B B Tcr Ampr Ampr X B ori ori Ampicillin resistant? yes yes Tetracycline resistant? No yes

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 screening Screening by insertional inactivation of the lacZ gene Lac promoter MCS (Multiple cloning sites, 多克隆位点） Ampr pUC18 (3 kb) lacZ’ ori 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 transformants Recombinant plasmid containing inserted DNA: white transformants Recreated vector (no insert) Recombinant plasmid (contain insert) back

Multiple cloning sites Multiple 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 KpnI SmaI XmaI BamHI XbaI SalI HincII AccI PstI SphI Lac Z

A plasmid vector for gene expression Expression vectors: allowing the exogenous DNA to be inserted, stored and expressed. Promoter and terminator for RNA transcription are required. Intact ORF and ribosomal binding sites (RBS) are required for translation. Include：(1) bacterial expression vectors, (2) yeast expression vectors, (3) mammalian expression vector

An bacterial expression vector T7 promoter RBS Start codon MCS Ampr Transcription terminator T7 expression vector ori An bacterial expression vector

A yeast expression vector MCS A yeast expression vector

Bacteriophage vector Two examples: λ phage bacteriophageλ λ replacement vector M13 phage M13 phage vector Cloning in M13 Hybrid plasmid-M13 vectors

λ phage 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)

Cloning large DNA fragments (Eukaryotic Genome project) 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. Genomic library VS cDNA library

Cosmid vectors Utilizing the properties of the phage l cos sites in a plasmid vector. A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the l head. The insert can be 37-52 kb

Formation of a cosmid clone Digestion Ligation C) Packaging and infect

(yeast artificial chromosome) YAC vectors (yeast artificial chromosome) 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.

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 YAC 2. Capacity is 300-350 kb 3. One to two copies in each cell 4. Easy to handle 5. More popular in genomic mapping

I1 Genomic libraries I1-1 Representative gene libraries Gene libraries and screening I1 Genomic libraries I1-1 Representative gene libraries I1-2 Size of library I1-3 Genomic DNA I1-4 Vectors

(made from cDNA- copy of mRNA) I1 Genomic libraries 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 (made from genomic DNA) Gene library cDNA libraries (made from cDNA- copy of mRNA)

--- Contain all the original sequences I1 Genomic libraries I1-1 Representative gene libraries --- Contain all the original sequences Missing original sequence Certain sequences have not been cloned. Example: repetitive sequences lacking restriction sites Too long for the vector used 2. Library does not contain sufficient clones

I1-2 Size of library (ensure enough clones) 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: ln (1-P) N = 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)] ln(1-0.99) Nhuman= = 6.9 ×105 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.

I1-3 Genomic DNA libraries I1 Genomic libraries I1-3 Genomic DNA libraries eukaryotes Purify genomic DNA Fragment this DNA : physical shearing and restriction enzyme digestion prokaryotes Clone the fragments into vectors

Eukaryotes :prepare cell nuclei 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 : Eukaryotes :prepare cell nuclei remove protein, lipids and other unwanted macro- molecules by protease digestion and phase extraction. Prokaryotes :extracted DNA directly from cells

pipeting, mixing or sonicaion 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.

Selection of restriction enzyme I1 Genomic libraries Selection of restriction enzyme Ends produced (sticky or blunt) & The cleaved ends of the vector to be cloned Sau3A: 5’-/GATC-3’, less selectivity BamH1: 5’-G/GATCC Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.

I1-4 Vectors Vectors Plasmid phageλ cosmid YAC I1 Genomic libraries 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

λ phage vector in cloning Long (left) arm short (right) arm cos Exogenous DNA (~20-23 kb) cos Long (left) arm short (right) arm cos cos Exogenous DNA (~20-23 kb)

λ replacement vector cloning 0.preparation of arm and genomic inserts 2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes Ligation 3. Infection and formation of plaques Library constructed

I 2 cDNA libraries Gene libraries and screening I2-1 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

No cDNA library was made from prokaryotic mRNA. I 2 cDNA libraries cDNA libraries No cDNA library was made from prokaryotic mRNA. Prokaryotic mRNA is very unstable Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.

cDNA libraries are very useful for eukaryotic gene analysis I 2 cDNA libraries cDNA libraries 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 expression of genes)

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. AAAAAAAAAAn 5’ cap

I 2 cDNA libraries

Three methods to isolate mRNA. I2 cDNA libraries Three methods to isolate mRNA. 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

I2-2 Check the mRNA integrity I2 cDNA libraries I2-2 Check the mRNA integrity 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 translated Analysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels

Enrichment: carried out by hybridization 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 gels Enrichment: carried out by hybridization Example: clone the hormone induced mRNAs (substrated cDNA library)

Second strand synthesis: best way of 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 Fig 1.1 The first strand synthesis mRNA 5’ AAAAA-3’ HO-TTTTTP-5’ Reverse transcriptase Four dNTPs mRNA 5’ AAAAA-3’ 3’ TTTTTP-5’ cDNA Terminal transferase dCTP mRNA 5’ AAAAA-3’ 3’-CCCCCCC TTTTTP-5’ cDNA Alkali (hydrolyaes RNA) Purify DNA oligo(dG) 5’-pGGGG-OH 3’-CCCCCCC TTTTTP-5’ cDNA Klenow polymerase or reverse Transcriotase Four dNTPs 5’-pGGGG -3’ 3’-CCCCCCC TTTTTP-5’ Duplex cDNA Fig 1.1 The first strand synthesis

Single strand-specific nuclease Duplex cDNA 5’-pGGGG -3’ 3’-CCCCCCC TTTTTp-5’ Single strand-specific nuclease 5’-pGGGG -3’ 3’-CCC TTTTTp-5’ Klenow polymerase treat with E.coRI methylase 5’-pGGGG -3’ 3’-CCCC TTTTTp-5’ Add E.colRI linkers using T4 DNA ligase HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH HO-CCGAATTCGGGGGG CCGAATTCGG-3’ 3’-GGCTTAAGCCCCCC TTTTTGGCTTAAGCC-OH E.colRI digestion 5’-pAATTCGGGGGG CCG-3’ 3’-CCCCCCC TTTTTGGCTTAAp-5’ Ligate to vector and transfom Fig2.1 Second strand synthesis

I2-5 Treatment of cDNA ends I2 cDNA libraries I2-5 Treatment of cDNA ends Blunt 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) Tailing with terminal transferase or using adaptor molecules Restriction enzyme digestion (E.coRI )

I2-6 Ligation to vector The process : I2 cDNA libraries Any vectors with an E.coRI site would suitable for cloning the cDNA. The process : Dephosphorylate the vector with alkaline phosphatase Ligate vector and cDNA with T4 DNA ligase (plasmid or λ phage vector)

I3 Screening procedures Gene libraries and screening I3 Screening procedures I3-1 Screening I3-2 Colony and plaque hybridization I3-3 Expression screening I3-4 Hybrid arrest and release I3-5 Chromosome walking (repeat screening)

I3-1 Screening I3 Screening procedures The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library . Using nucleic acid probe to screen the library based on hybridization with nucleic acids. Analyze the protein product.

I3 Screening procedures Screening libraries Searching the genes of interest in a DNA library Hybridization to identify the interested DNA or its RNA product Radiolabeled probes which is complementary to a region of the interested gene Probes: An oligonucleotide derived from the sequence of a protein product of the gene A DNA fragment/oligo from a related gene of another species Blotting the DNA or RNA on a membrane Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane

I3-2 Colony and plaque hybridization I3 Screening procedures I3-2 Colony and plaque hybridization Transfer the DNA in the plaque or colony to a Nylon 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) (Alkali treatment) X-ray film(radio- actively labeled ) antibody or enzyme (modified nucleotide labeled Wash to remove unhybri- dization probe and visualize Line up the hybridizated region or repeated hybridization

I3 Screening procedures Transfer to nitrocellulose or nylon membrane Keep master plate Select positive from master plate Denature DNA(NaOH) Bake onto membrane Probe with 32p-labled DNA complementary to gene of interest Expose to film Screening by plaque hybridization

I3-3 Expression screening I3 Screening procedures I3-3 Expression screening Identify the protein product of an interested gene Protein activity Western blotting using a specific antibody

Expression screening (1) I3 Screening procedures 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).

Expression screening (2) I3 Screening procedures Expression screening (2) Antibodies can be used to screen the expression library. 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

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 Overexpression in cells 超表达，观察表型 RNAi 干扰技术 Yeast two hybrid system 酵母双杂交等技术寻找与目的基因相关的蛋白 Protein expression and antibody preparation表达蛋白与抗体制备 Localization of protein 蛋白在细胞中的定位

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