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[II] Molecular Techniques for Studying Gene Expression Basics of recombinant DNA technology Methods used to monitor the expression of genes RT-PCR vs. Real-time RT-PCR DNA microarray analysis Transgenic analysis and gene inactivation Chromatin immunoprecipiotation Methods of analysis of proteins Reading List II Cloning and Characterizing DNA Molecule **What is a recombinant DNA molecule? **Cloning of a genomic gene **Cloning of a complementary DNA (cDNA) Why is it necessary to clone genes? • Naturally occurring DNA molecules are very long and a single molecule usually carrying many genes • • • • Genes may occupy only a small proportion of the chromosomal DNA and the rest are noncoding sequences (a human gene might constitute only 1/1,000,000 of a chromosomal DNA) Unlike enzymes, there is no convenient method to identify a gene. Therefore, it is impossible to obtain a sizable amount of DNA encoding a gene by the ordinary purification method It is impossible to purify individual mRNA due to lacking method to identify individual mRNA species. Clone individual cDNA of the mRNA is the only solution Technological Breakthrough in Molecular Biology Leading to Cloning of DNA • • • • Discovery of restriction enzymes & modification enzymes --allowing cutting and manipulation of DNA molecules Methods of isolating DNA molecules and agarose gel electrophoresis for separating DNA molecules Plasmids and bacteriophage as vectors for in vivo amplification of DNA molecules Methods of introducing DNA molecules into cells --“Transformation” • Hybridization method for identifying specific DNA molecules • Reading List: Restriction enzyme – a background paper Recombinant DNA and gene cloning General Strategy of Recombinant DNA Technology • • Recombinant DNA technology: cloning and manipulation of DNA molecules (genomic DNA or cDNA) Vector + DNA fragment Recombinant DNA Replication of recombinant DNA molecules in host cells Isolation, sequencing, and manipulation of purified DNA fragment Several Enzymes that Digest DNA Molecule • • • • (a): Terminal phosphatase (b): Nuclease: digest either the first ester bond or the second ester bond (c): Endonuclease (d): Exonuclease Restriction Enzyme (I) • • • • • Restriction enzymes were originally discovered in bacterial cells serving to remove invading foreign DNA, but bacterial DNA itself can be protected from restriction enzyme digestion by adding methyl group (-CH3) to A or C on the DNA Restriction enzymes were discovered by W. Arber, D. Nathans and H. Smith in 1960’s. They were awarded with a Nobel Prize in 1978 DNA fragments generated by digestion with some restriction enzymes are with sticky ends Restriction enzymes have been used to prepare DNA molecules for cloning Reading Assignment: Nobel lecture by H. Smith 1978 • Restriction Enzymes (II) Restriction enzymes: Enzymes from bacterial cells that can cut DNA molecules at specific nucleotide sequence Restriction site (palindromic sequence) • • Some enzymes digest DNA to produce sticky ends while others produce blunt ends Palindromic sequence; 4 cutters = 44 (256 bases); 6 cutters = 46 (4096 bases) Restriction Enzymes (III) (a). Some restriction endonucleases that cleave the restriction sites to generate a staggered cut (b). Other restriction endonucleases that cleave the restriction sites to generate blunt ends Restriction Map of a DNA Fragment • • • • A restriction map of a DNA fragment is a linear sequence of sites separated by defined distances on DNA The map shown above identifies three restriction sites cleaved by restriction enzyme A and two sites by restriction enzyme B Thus DNA fragment digested by restriction enzyme A alone generates 4 fragments and digested by restriction enzyme B alone generates 3 fragments These DNA fragments can be resolved by electrophoresis on an agarose gel and their determined DNA Modification Enzymes • DNA Modification Enzymes: Enzymes that can modify DNA molecules DNA Ligase: An enzyme that can ligate two DNA molecules together by making a phosphodiester bond DNA polymerase I: involves in synthesis of DNA molecules (identified by Athur Kormberg) DNA phosphorylase: An enzyme that can remove phosphate group from DNA molecules DNA kinase: An enzyme that can add a phosphate group onto the 5’end of a DNA molecule Terminal transferase: An enzyme that can add nucleotide on to 3’-end of the DNA molecule Endo- and exo-nucleases: break a phosphodiester bond Reverse transcriptase: make a strand of DNA by copying RNA template (identified by H. Temin and D. Baltimore independently) Tqa polymerase: synthesize DNA under high temperature. This enzyme is isolated from Thermus aquatica (identified by T.D. Brock) Cloning Vectors (I): Basic Components of a Bacterial Plasmid Cloning Vector • • • Plasmid cloning vector in E. coli is a circular DNA molecule of 1.2 to 3 kb Plasmid Vectors contain: Selectable gene such as ampr Replication origin (ORI) A synthetic polylinker with unique restriction enzyme recognition site DNA fragment from a few base pairs to about 10 kb can be inserted into the cloning vector and manipulated Construction of a Recombinant Plasmid • • • • • Replication origin Selection marker: Ampicillin resistant gene or others MCS: mutiple cloning site DNA fragment of interest can be inserted at MCS for amplification DNA fragment smaller than 10 Kb can be amplified in a bacterial plasmid Cloning Vectors (II): Cloning Vectors for Cloning Genomic DNA (the earliest version) 1. A Phage Cloning Vector: Lambda (l) phage DNA has been developed into a cloning vector for cloning DNA fragment up to 20 kb. This is the earliest type of vector used to construct a genomic library Recombinant l Phage Cloning Vectors (III): Cosmid and Phagemid • • Cosmid is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequence. Cosmids (cos sites + plasmid = cosmid) DNA sequences are originally from the Lambda phage. Cosmids can be used to build genomic library. A phagemid is developed as a hybrid of the filamentus phage M13 and plasmid to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. It contains an ORI for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Cloning Vectors (IV): Cloning vectors for cloning a large piece of DNA • Bacterial artificial chromosome (BAC): A plasmid contains a functional fertility factor, replication origin, partition factor, selection factor and T7 and Sp6 promoters This plasmid (Fplasmid) can accommodate DNA fragment of about 100300 Kb The recombinant plasmid can be introduced into E. coli cells by electroporation Cloning Vectors (IV): Cloning vectors for cloning a large piece of DNA • Yeast Artificial Chromosome: This cloning vector contains Tel (telomere), CEN (centromere) and ORI (replication origin) of yeast cells. It can accept a large piece of DNA (100 kb to 3,000 kb) The recombinant cloning vector is introduced into yeast cells by electroporation for amplification Cloning Vectors for Different Purposes • Detection of Nucleic Acids Agarose gel electroporation Hybridization, Southern blot and RNA northern blot analyses Agarose Gel Electrophoresis to Separate DNA • Agarose is an inert carbohydrate isolated from seaweeds. DNA molecules of different sizes can be separated in an agarose gel by electrophoresis and visualized by staining the gel with ethedium bromide & observe under UV light. The DNA fragment can be recovered from the gel by extraction with phenol and chloroform DNA Fragments Visualized Under UV Light DNA on agarose gel is stained with ethedium bromide and observed under UV light Sizes of DNA Fragments Determined by Agarose Eectroporation Separation of Molecules by Gradient Centrifugation • • • Macromolecules (DNA, RNA and proteins) can be separated by their sizes or densities Macromolecules of different sizes can be separated by electrophoresis on agarose or polyacrylamid gels Macromolecules of different densities can be separated by grant centrifugation Sedementation centrifugation Equilibrium centrifugation Hybridization • Hybridization: Two fragments of single-stranded homologous DNA molecules can form hybrid through hydrogen-bonding formation (base pairing) 5’-GTACTTAGGCAATTGGGCA-3’ 3’-CATGAATCCGTTAACCCGT-5’ • If one of these two strands of DNA is labeled with • • radioactive isotopes, the hybrid will be easily visualized by autoradiography Hybridization can occur between two homologous DNA molecules or a DNA molecule and a RNA molecule “Hybridization” has been used in Southern blot and RNA northern blot analyses Southern Blot Hybridization This method is developed by Edwin Southern Results of Southern Blot Hybridization Fluoerescence in situ Hybridization This technique is used for cytological localization of molecules in the cell Methods of Introducing DNA into Cells • Recombinant DNA molecules can be transferred into bacterial cells, plant cells and animal cells by transformation methods: Bacterial cells: regular transformation method or electroporation method Plant cells: Agrobacterium infection, electroporation or particle gum bombardment Animal cells: microinjection, Ca3(PO4)2 precipitation method, lipofection or electroporation method Electroporators Electroporators can generate high frequency pulses to create transient opening on cells by which the DNA molecules enter into the cell • Cloning of Genomic DNA and cDNA Complementary (c) DNA Verses Genomic Gene Complementary DNA (cDNA): A reverse sequence of an mRNA, synthesized by using mRNA as a template and reverse transcriptase as the enzyme to catalyze the reaction Genomic Gene: A gene contains the coding region (both intron and exon) and the control region Why should one clone a cDNA or a genomic gene? Purposes of Cloning Genomic DNA and cDNA • Genomic DNA: Studying structures of genes, gene families Identifying promoters and other regulatory elements Studying evolution of genes • cDNA: Studying structures of mRNA Measuring the levels of mRNA Studying developmental stage-specific and tissue-specific expression of genes Studying processing and stability of mRNA Producing recombinant proteins Construction of Genomic DNA Library • • • Since the sizes of genomic genes vary from several kbs to several hundred kbs, bacterial plasmid vector is not very suitable for cloning a complete gene Commonly used vectors for cloning complete genes are: Lambda phage vector (up to 20-25 kb) Artificial bacterial chromosome (BAC) vector (up to 500 kb): it contains a bacterial origin of DNA replication and genes required to regulate their own replication Yeast artificial chromosome (YAC) vector (up to 1000 kb): it contains all the elements of yeast chromosome i.e., elements for replication of the chromosome during S phase, segregation of chromosome, telomeres, genes for selection and DNA fragment to be cloned Genomic DNA library: A collection of DNA fragments of a genome Isolation of Genomic Genes from DNA Libraries • • Isolating gene clones from libraries by colony hybridization or plaque hybridization Characterizing the inserted DNA by: Restriction enzyme digestion (establishing restriction maps) Determining the nucleotide sequence of the inserted DNA Expressing the gene product in E. coli or mammalian cells Colony Hybridization • • This method is based on the principle that two homologous strands of nucleic acids can form hybrid form If one of the strands of nucleic acid is radio-labeled, the hybrid can be visualized by autoradiography Plaque Hybridization • This technique is suitable for isolating genes cloned in a lambda phage cloning library Restriction Enzyme Mapping • • Restriction enzyme recognition sites can be used to make maps of DNA --- restriction mapping Restriction map of a cloned DNA is made by digesting the cloned DNA with a series of restriction enzymes and determine their sites on the DNA Restriction map of the catfish growth hormone gene Structure of Dideoxynucleotide Triphosphate • • The nucleotide sequence of a piece of DNA can be determined by: Chemical sequencing method developed by Maxam Gilbert Dideoxy chaintermination method developed by F. Sanger Incorporation of dideoxynucleotide triphosphate into the going chain of DNA will stop the elongation of the DNA chain A Single Strand DNA to be Sequenced Assigned Reading: Nobel lecture by F. Sanger on “DNA sequencing”, 1980 Separating the Products on a Denaturing Polyacrylamid Gel 3’ Separating the Products on an Automated Sequencing Machine (1.8 x 106) (1.2 x 107) (9.7 x 107) (1.0 x 108) (1.8 x 108) (3.2 x 109) Strategies of Assembling Whole Genome Sequence Chromosome Walking • • This technique allows the isolation of a long eukaryotic gene An alternative is to construct a BAC library that contains long piece DNA molecules Making cDNA from an Eukaryotic Gene • • To express an eukaryotic gene into its product in bacterial cells, the mRNA is converted into cDNA The cloned cDNA can be expressed into protein product by inserting it into a plasmid containing a functional promoter (expression vector) Construction of a cDNA Library • • • • Synthesis of 1st strand cDNA Synthesis of double strand cDNA Ligate the double stranded cDNA into a plasmid vector or l phage vector Propagate the library in E. coli cells Lambda Phage Cloning Vector for Cloning cDNA 1. Phage Cloning Vector: Lambda (l) phage DNA has also been developed for cloning cDNA as well. This type of vector is suitable for cloning cDNA banks. In this case, the non-essential region in the lambda phage genome is not removed. If a functional promoter is present in the mutiple cloning site, the cloned cDNA will also express into it gene product cDNA Library (Bank) • • Lambda gt10: for cloning regular cDNA that can be identified by hybridization Lambda gt11: for cloning cDNA that can express the protein product, so the cDNA clone can be identified by immunoblotting Expression Vectors • Expression vector: A cloning vector that contains a “functional promoter”, if this vector also contains a gene that its expression can be easily seen (detected), it is said that this vector contains a “reporter gene” Leuciferase gene derived from fireflies is a popular reporter gene. An expression vector containing leuciferase gene can be used to define the functional promoter and other regulatory sequence Expression of a lacZ gene stained for b-galactosidase in a mouse embryo Visualization of Cancer Cell Growth by GFP • • • External and internal images of bone metastasis of AC3488 GFP (A) External images of tumors in the skeletal system [including the skull (arrow heads), scapula (thick arrows), spine (fine arrows), and liver metastasis (largest arrows) in a dorsal view of live, intact nude mouse (B–I) Series of external fluorescence images of metastatic lesions in the skull, ribs, spine, and tibia, (B, D, F, and H) compared with corresponding images of the exposed skeletal metastases (C, E, G, and I) (Bars =1280 mm). • Methods used to monitor the expression of genes Using Cloned DNA Fragments to Study Gene Expression RNA northern blot analysis In situ hybridization RNase protection assay Measuring mRNA levels by RT-PCR or real-time RT-PCR assay Measuring the profiles of gene expression by DNA microarray Measuring the profiles of gene expression by RNA sequencing analysis Using Cloned DNA Fragments to Study Gene Expression • • • • Hybridization techniques permit detection of specific DNA fragments and mRNAs Southern blotting method: DNA-DNA hybridization RNA northern blotting method: RNA-DNA hybridization, qualitative and semi-quantitative methods In situ hybridization RT-PCR DNA microarrays can be used to evaluate the expression of many genes at one time E. coli expression systems can produce large quantities of proteins from cloned genes Cloned genes can also be introduced into yeast cells and other animal cells for production of large quantities of proteins for analysis DNA Southern Blot & RNA Northern Blot Techniques RNA RNA Northern Blot Analysis of b-Globin mRNA • • • Total RNA was extracted from erythrolukemia cells grown but not induced and in cells induced to stop growing and allowed to differentiate for 48 h or 96 h The RNA samples were resolved in agarose gels and transferred to a nylon membrane The membrane was hybridized to radiolabeled b-globin cDNA In Situ Hybridization to Detect of the mRNA of Sonic Hedgehog in Mouse Embryos Whole embryo (10 days old) A section of the embryo Drosophila Embryo In this experiment, the in situ hybridization technique is used to localize the site of expression of sonic hedgehog gene in mouse embryos Principle of RNase Protection Assay AAAAAAA mRNA + 32P DNA Hybridization and digested with RNase The product is analyzed on a ureapolyaccrylamid gel RNase Protection Assay to Measure IGF-I mRNA Levels in the Liver of Rainbow Trout CPM or OD600 If a standard curve is established using known quantities of cRNA, the amount of the unknown can be determined mg cRNA Nuclear Run-on Transcription Assay • • • • • Nuclear run-on assay can be used to ascertain which gene is active in a given cell allowing transcription to continue in isolated nuclei Specific transcript can be identified by their hybridization to known DNAs on dot blot It can also be used to determine the effects of assay conditions on nuclear transcription Transcription activity of a specific gene can be determined It can also be used to measure template activity Nuclear Run-On Assay to Measure the Transcription Rates of Genes in Various Tissues Measuring levels of mRNA by RT-PCR or realtime RT-PCR assay • • Principle of Reverse Transcriptioin (RT)PCR End Point RT-PCR: Measuring the presence of a particular mRNA Can be adapted to perform semi-quantitative determination of an mRNA Real-time RT-PCR: For quantitative measurement of the level of an mRNA Absolute quantitative RTPCR Comparative quantitative RT-PCR PCR Amplification Program • • 1 cycle at 95 oC for 3 min to denature all templates 40-50 cycles: 95 oC for 15 sec for denaturation of PCR products At annealing temperature for 15 sec to anneal primers to the templates 72 oC for 30 sec for synthesis of DNA Determine Levels of mRNA by Semiquantitative RT-PCR • • • If levels of b-actin mRNA in various tissues are constant, then the levels of mRNA of interest can be expressed in terms of relative to levels of b-actin This assumption will only work when the increase of levels of mRNA is in the linear range of the detection Example given in the left of this slide is showing how the levels of NPY mRNA is determined by this method Competitive Quantitative RT-PCR AAAA IGF-II mRNA to be determined In vitro transcription Competitor mRNA Reverse Transcription Competitor cDNA Reverse Transcription IGF-II cDNA Various amounts of both templates and fixed amount of amplification primers and proceed PCR amplification Competitive Quantitative RT-PCR When the ratio of target/competitor equals to 1, the levels of competitor equals the level of the target Assigned Reading: Greene and Chen, 1999. Quantitation of IGF-I, IGF-II, and multiple insulin receptor mRNAs during embryonic development in rainbow trout. Molecular Reproductive Development 54: 348-361. Real-time PCR Machine Principle of Real-Time RT-PCR • • • SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. SYBR Green I binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = 497 nm) and emits green light (λmax = 520 nm). The stain preferentially binds to double-stranded DNA, but will stain single-stranded DNA with lower performance. SYBR green can also stain RNA with a lower performance than DNA. Since double stranded DNA can interact quantitatively with SYBR Green 1 preferentially and thus can be used to monitor the amount of DNA synthesized after each cycle of PCR reaction. Cycle threshold (CT): CT is defined as the number of PCR cycles required for the fluoescent signal to cross the threshold (i.e., exceeds background level). This is usually determined automatically by the real-time PCR machine set in the factory Determination of Cycle Threshold (CT) Sample Set Up in a 96-Well Plate 1 2 3 4 5 6 7 8 9 10 Unkn-1 Unkn-1 Unkn-1 Unkn-5 Unkn-5 Unkn-5 Unkn-9 Unkn-9 Unkn-9 82.20 82.00 82.00 82.20 82.00 82.00 82.00 82.00 82.00 Content Sample A Peak 1 Peak 2 Content Sample B Peak 1 Peak 2 None None None None None None None None None Content Unkn-2 Unkn-2 Unkn-2 Unkn-6 Unkn-6 Unkn-6 Unkn-10 Unkn-10 Unkn-10 Peak 1 82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00 Peak 2 None None None None None None None None None Content Unkn-3 Unkn-3 Unkn-3 Unkn-7 Unkn-7 Unkn-7 Unkn-11 Unkn-11 Unkn-11 Peak 1 82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00 Peak 2 None None None None None None None None None Content Unkn-4 Unkn-4 Unkn-4 Unkn-8 Unkn-8 Unkn-8 Unkn-12 Unkn-12 Unkn-12 Peak 1 82.20 82.00 82.00 82.00 82.00 82.00 82.00 82.00 82.00 Peak 2 None None None None None None None None None Content NTC-1 NTC-1 NTC-1 Peak 1 None None None Peak 2 None None None Sample C Sample D Sample E Sample F Content Sample G Peak 1 Peak 2 Content Sample H Peak 1 Peak 2 11 12 PCR Amplification Program • • 1 cycle at 95 oC for 3 min to denature all templates 40-50 cycles: 95 oC for 15 sec for denaturation of PCR products At annealing temperature for 15 sec to anneal primers to the templates 72 oC for 30 sec for synthesis of DNA Melting Profile of RT-PCR Products • From the melting profile, one can determine whether the PCR product is amplified from a single target Absolute Quantitative Real-time RT-PCR • Construction of a standard curve: Construct a synthetic cRNA of your interest Reverse transcribe the cRNA into cDNA Set up Real-time PCR with known and increasing amount of cDNA Plot Ct value vs. starting quantity of cDNA Levels of Gene Expression Determined by Absolute RT-PCR NKX2.5 Norm. GI,GII Norm. Norm. GI,GII β-actin GATA 5 GI,GII Norm. Norm. GI,GII GI,GII Norm. GI,GII Assigned Reading: Chun et al., 2006. Trout Ea4- or human Eb-peptide of proIGF-I disrupts heart, red blood cell, and vasculature development in zebrafish embryos Levels of Gene Expression Determined by Absolute RT-PCR GATA 2 GATA 1 Norm. GI,GII Norm. GI,GII Norm. Norm. Norm. GI,GII VEGF GI,GII GI,GII Norm. GI,GII Reduction of mRNA Levels of Heart, Vasculature and Blood Cell Development-Related Genes in rtEa4Peptide Injected Embryos Molecules of mRNA/embryo at 36 hpf Gene Normal Defective embryos Fold of reduction NKX2.5 2.77±2.04X106 8.02±5.96X103 242.1±1.0 GATA5 3.94±1.07X105 2.41±6.58X104 11.7±0.9 GATA1 1.63±0.46X106 2.27±0.12X105 5.5±1.2 GATA2 1.37±0.22X104 3.59±0.76X103 2.7±1.0 VEGF 2.87±0.96X105 3.09±0.41X104 6.4±1.1 β-actin 5.77±1.19X106 4.16±1.33X106 1.0±1 .0 Relative Quantitative RT-PCR Calculation of Relative Expression Levels ∆CT= CT (target) – CT (normalizer) ∆∆CT = ∆CT (treated) - ∆CT (control) Fold =1/[2-(∆ ∆ CT)] GAPDH: glyceraldehyde-3-phosphate dehydrogenase or b-actin mRNA is usually used as normalizer Asigned Reading: Chen et al., 2007. Suppression of growth and cancerinduced angiogenesis of aggressive human breast cancer cells (MDA-MB-231) on the chorioallantoic membrane of developing chicken embryos by Epeptide of pro-IGF-I Comparative Real-Time PCR Analysis of Genes Up- and Down-Regulated by E-Peptide Names of Genes Relative Expression Levels uPA PAI 1 BCL 2 Cysteine-rich angiogenesis inducer Tumor-associated Ca++ signal inducer TYPO3 protein tyrosine kinase Tumor rejection antigen (Gp96) Heat shock protein 90 KDa protein 1a Heat shock 70 KDa protein 10 Capase 3 Fibronectin 1 Laminin receptor 1 0.52 + 0.10 0.42 + 0.04 0.28 + 0.04 0.86 + 0.12 0.44 + 0.12 3.02 + 0.80 3.46 + 0.96 3.40 + 0.35 2.92 + 0.39 4.58 + 0.35 2.32 + 0.31 1.41 + 0.11 *Fold of reduction = 2-(∆ ∆ CT), where ∆ ∆ CT is ∆ CT sample - ∆CT control where ∆ CT = CT (Target) – CT (normalizer) • Microarray: a technique that allows you to determine the expression of many genes at one time Why DNA Microarray Technology? • Gene discovery: Examples • Disease diagnosis: • Drug discovery: Pharmacogenomics Profiling of cancer-specific expressing genes Tissue-specific expression of genes Developmental-specific expression of genes Collections of genes showing expression of genes specific to certain types of diseases Examples: Specific cancer type, hematopoietic disease etc. To find correlations between therapeutic responses to drug and gene profiles of patience Toxicological research: Toxicogenomics • To find correlations between toxic responses to toxicants and changes in the gene profiles of the objects exposed to such toxicants Principle of DNA Microarray • • • • Genes (cDNA or oligonucleotides are spotted on glass plates Messenger RNA is reverse transcribed into cDNA and labelled with Cy3 (emission 570nm, green) or Cy5 (emission 670 nm, red) Hybridization (mixing Cy3-cDNA and Cy5 cDNA) Scan the slide to detect the hybridization signals Enlarged Photo of a Microarray Chip • • • This array has 2400 human genes Red indicates increase of expression; yellow equal expression and green reduce of expression This technique can help to determine the profiles of gene expression Interpreting the Scanned Image • • • • The measured intensities from the two fluorescent reporters have been false-colored red and green and overlaid Yellow spots have roughly equal amounts of bound cDNA from each cell population and so have equal intensity in the red and green channels (red + green = yellow) Spots whose mRNA’s are present at a higher level in one or the other cell population show up as predominantly red or green The ratio of fluorescent intensities for a spot is interpreted as the ratio of concentrations for its corresponding mRNA in the two cell populations Example Novel Anti-cancer Activity of ProIGF-I E-Peptide Assigned Reading: Chen et al., 2007: Suppression of Growth and CancerInduced Angiogenesis of Aggressive Human Breast Cancer Cells (MDA-MB-231) on the Chorioallantoic Membrane of Developing Chicken Embryos by E-peptide of Pro-IGF-I Insulin-Like Growth Factor (IGF) I Primary translation product of IGF-I B S C A D E Post-translational processing Signal peptide S B C A Mature IGF-I D E E-peptide E-Peptide of Pro-IGF-I • The pro-hormone peptide of IGF-I • It contains about 77 amino acid residues • Presents in fish, human and other animals • It is highly soluble in aquaous buffers • Can be readily prepared by expressing the gene in E. coli and other cell systems Novel Anti-Cancer Activities of the E-Peptide • • • • • Exerts dose-dependent mitogenic activities in established non-transformed cell lines (Tian et al., 1999) Induces morphological differentiation and inhibits anchorage-independent growth in oncogenic transformed cell lines (Chen et al., 2002; Kuo and Chen, 2002) Inhibits tumor cell growth and invasion, and tumorinduced angiogenesis in developing chicken embryos (Chen et al., 2007) Induces programmed cell death in cancer cells (Chen et al., 2011) Up-regulate fironectin 1 and laminin receptor genes and down-regulate uPA, tPA and TIMP1 (Siri and Chen 2006a, 2006b) Is There Molecular Evidenced • • If E-peptide of IGF-I can control cancer cell growth, there must be genes that are up- or down-regulated by E-peptide Using microarray analysis technique, we determined genes related to cancer cell activities that are up- or down regulated by e-peptide in aggressive human breast cancer cell (MBA-MD-231) Isolate RNA samples from MBA-MD-231 cells treated wit Epeptide and untreated cells Prepare cDNA from both RNA smples Labele E-peptide treated cDNA with Cy3 and the untreated cDN with Cy5 Combine both labeled cDNA and hybridized to a high density human gene chip and scan the results Results of the Microarray Assay • • • • From repeat screening a gene chip contain 10,000 human genes, more than 1,000 genes were found to be up- or down-regulated by E-peptide to a different degree Those genes with ratio of treated/untreated signals over + or – 2 are considered real difference Examples of up-regulated and down-regulated genes are summarized in the next two slides To confirm the microarray results, the mRNA levels of each gene in the treated and untreated RNA samples should be determined by real-time quantitative or comparative RT-PCR Some Genes Down-Regulated by E-Peptide • • • • • • Oncogenes: TC 21, P53, vav 1, v-myb, c-H-ras (p21), sarc, v-erb 2 (high expression in tumors) Angiogenesis: Cysteine-rich angiogenic inducer 61 IGF-II, IGF-IIR, IGFBP-3, -4 & -7 Cell adhesion: Catenin a-like-1, Integrin a-3, Integrin b3, Integrin b-4, Tight junction protein 1 Proteases: Cathepsin Z, Plasminogen activator inhibitor type I, Urokinase plasminogen activator, Tissue plasminogen activator Cell cycle, growth & proliferation: Cyclin-independent kinase inhibitor 1A, Latent transforming growth factor b-binding protein 3, Serine inducible kinase, Colony stimulating factor 1, Thymosin b-4 Cytoskeleton molecules: Keritin-7, -8, and -18 Some Genes Up-Regulated by E-Peptide • • • • • • Cell adhesion: Fibronectin 1, Laminin receptor 1 Cytoskeleton molecules: Keratin-1, Restin Proteases and inhibitors: Tissue inhibitor of metalloprotease I, Cathepsin C, Zinc metalloprotease Cell cycle, growth & proliferation: Cyclin A2, B1, B2, Cyclin-dependent kinase 7, Topoisomerase II a Cell signal transduction molecules: TyrO3 protein kinase, Mitogen-activated protein kinase kinase 3, csar tyrosine kinase, Phosphotidylinositol-4-phosphate5 kinase type Ia Tumor rejection antigen (gp96) 1, Cdc 14 phosphatase, Protein tyrosine kinases, Heat shock proteins 70 and 90 Comparative Real-Time PCR Analysis of Genes Up- and Down-Regulated by E-Peptide Names of Genes Relative Expression Levels uPA PAI 1 BCL 2 Cysteine-rich angiogenesis inducer Tumor-associated Ca++ signal inducer TYPO3 protein tyrosine kinase Tumor rejection antigen (Gp96) Heat shock protein 90 KDa protein 1a Heat shock 70 KDa pr5otein 10 Capase 3 Fibronectin 1 Laminin receptor 1 0.52 + 0.10 0.42 + 0.04 0.28 + 0.04 0.86 + 0.12 0.44 + 0.12 3.02 + 0.80 3.46 + 0.96 3.40 + 0.35 2.92 + 0.39 4.58 + 0.35 2.32 + 0.31 1.41 + 0.11 RNA-Seq Technology RNA-seq, also called "Whole Transcriptome Shotgun Sequencing" ("WTSS"), refers to the use of high-throughput technologies to sequence cDNA or RNA in order to get information about a sample's RNA content. The technique has been adopted in studies of diseases like cancers. With deep coverage and base-level resolution, this technology provides information on differential expression of genes, including gene alleles and differently spliced transcripts; non-coding RNAs; post-transcriptional mutations or editing; and gene fusions Reading Assignment: 1. RNA-Seq 2. Wang et al., 2009. RNA-Seq: a revolutionary tool for Transcriptomics. Nature Review: Genetics 10: 57-63. • Transgenic technology Biotechnological application Assigned Reading : Application of recombinant DNA technology Gene Transfer in Plants Golden Rice Transfer genes that can make vitamin A into rice Transgenic Animal Technology • Transgenic Animals: Animals with foreign gene(s) integrated into their genomes • The first transgenic animal was produced in mice by Richard Palmiter and Ralf Brinster in early 1980s Transgene: Human growth hormone gene driven by the promoter of metallothionein gene Procedure for Producing Transgenic Mice Transgenic Aquatic Animals • • • We have been working on gene transfer technology since mid 1980s Over the past two decades, we have produced several speccies of transgenic aquatic animals by microinjection, electroporation, infection with pantropic retroviral vectors, and lipofection References: Zhang et al., MRD 25: 3-13, 1990. Lu et al., Mol Marine Biol. Biotech 1:366375, 1991. Lu et al., PNAS 93: 3482-3486. 1996. Lu et al., Mol Marine Biol Biotecnnol 7: 289295, 1997. Sarmasik et al., Marine Biotechnol 3: 465473, 2001 Sarmasik et al., Marine Biotechnol 3: S117S184, 2002 Lu et al., Marine Biotechnol 4: 328-337, 2002. Pore Formation on Bacterial Cell Membrane by Cecropin Cecropin Pores Formation Prototype of Transgene CMV IgG-SP Cecropin P1 Promoter Transfer the transgene construct into sperm of rainbow trout by electroporation Sperm-Mediated Gene Transfer Transgene Parameters: Dilution buffer, Dilution factor, DNA/sperm, Voltage, Pulse # Mortalities of F2 and F3 Cecropin P1 Transgenic Fish Challenged with A. salmonicida % Mortality (Mean ± S.D.) Families F2 Generation S8#Y419 S7#375 S9#746 S7#342 S8#505 S9#638 S9#659 U6#768 Non-transgenic 12 ± 3 14 ± 5 20 ± 6 30 ± 3 20 ± 4 10 ± 2 25 ± 6 80 ± 3 80 ± 6 F3 Generation 15 ± 5 18 ± 8 30 ± 5 35 ± 4 26 ± 8 14 ± 8 40 ± 10 79 ± 12 85 ± 8 For each family, challenge was conducted with 50 fish/family (0.4 to 0.5 g body weight) in triplicates and the dose of A. salmonicida used in each challenge study brings about 80% mortality in nontransgenic fish. Some Key Terms • Totipotent: Fertilized oocytes and blastomeres of 2, 4-, and 8-cell stage embryos. Could contribute to the development of the embryo proper and the placental tissue. • Pluripotent: Inner cell mass (ICM) of blastocysts, embryonic ectoderm and primordial germ cells of fetal stages. Could contribute to the development of embryo proper only. • Embryonic germ cells: Stem cells developed from primordial germ cells. Could develop into all somatic cell types of an embryo. These cells are also pluripotent. • Somatic stem cell: Stem cells could only develop into restricted cell types. (e.g. Skin) Stem cells: Stem cells are cells found in most, if not all, multicellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiation into a diverse range of specialized cell types. Two types of mammalian stem cells are: Embryonic stem cells: Isolated from the inner cell mass of blastocyst Adult stem cells: Found in adult tissues. Assigned Reading: Embryonic stem cells. Totipotent and Pluripotent Cells Different Paths of Generating Human ES Cells from In Vitro Fertilized Human Oocytes Inactivation of Genes by Knock-Out Method • • Gene knock-out: Interrupt a gene by inserting a small piece DNA into it To perform this technique, it requires: ES or EG cells Introduce the interrupting DNA into ES or EG cells and isolate recombinants Injecting the recombinant cells into blastoceal cavity of mouse embryos Transfer the embryos into pseudo-pregnant female mice Assigned Reading: Elimination of a gene by the knock out technology Construction of a Gene-Targeted Disruption Construct • • • • neor: a marker gene that confers resistant to G-418 tkHSV: a marker gene confers sensitivity to ganciclovir When the above construct is introduced into mouse stem cells, stem cells with gene been disrupted can be isolated The isolated ES cells can be used to generate gene knock out animals Isolation of Recombinant Stem Cells with a Gene Been Interrupted Production of Germ Line Knock Out Animals • • • • • Introduction of knock out heterozygous ES cells into the blastoceal cavity of mouse embryos Transfer embryos into pseudopregnant female mice Select chimeric mice Mat chimeric mouse with homozygous black mouse Select brown mice for homozygous knock out mice Knock Out Genes in Somatic Cells by the loxP-Cre Recombination System • • loxP site: site-specific DNA recombination site Cre: enzyme catalyzing recombination Inactivation of a Gene by Using RNAi • • In vitro or in vivo production of siRNA Inhibition of mex3 mRNA by injecting siRNA into the cells • Methods of analyzing proteins produced in different tissues or composition of proteins in different cells: SDS-polyacrylamide gel electroporation Immuno western blot analysis Two dimensional electroporation analysis Proteomic analysis 1 2 3 4 5 6 7 8 9 SDS Polyacrylamide gel stained with coomassie brilliant blue dye 25.9kd 19.4kd 14.8kd 6.0kd 1 2 3 4 5 6 7 8 9 SDS polyacrylamide gel stained with silver stain 25.9kd 19.4kd 14.8kd 6.0kd Silver staining 1 2 3 4 5 6 7 8 9 Immuno western blot analysis 25.9kd 19.4kd 14.8kd 6.0kd Anti Histag Antibody Immuno Western Blot Analysis Concept of Two Dimensional Gel Electrophoresis Comparison of Two Protein Samples Strategy of Sequencing a Protein by Mass-Spectrometry Identification of Proteins in Organelles by Gradient Centrifugation and LC-MS/MS Analysis Chromatin Immunoprecipitation This technique can be used to detect proteinDNA interactions in the native chromatin context in vivo. The associated DNA is purified for analysis by identifying its specific sequence by PCR or by labeling the DNA and applying to a tiling array to detect genome wide interactions Assigned Reading [II] 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Restriction Enzymes: a background paper Recombinant DNA and gene cloning Nobel lecture by H. Smith, 1978 Nobel lecture by O. Shimomura on discovery of GFP Nobel lecture by K. Mullis PCR, 1993 Nobel lecture by Sanger Nobel lecture by Michael Smith Elimination of a gene by the knock out technique Greene and Chen, 1999. Quantitation of IGF-I, IGF-II…….. Chun et al., 2006: Trout Ea4 and human Eb-peptide …….. Chen et al., 2007 Embryonic stem cells RNA-Seq RNA-Seq: a revolutionary tool for transcriptomics