Molecular Genetic Techniques PDF

& Molecular Genetic Techniques (5 of 6) s tru , gy al n, c. D tio.S of or h.D ell -24...

& Molecular Genetic Techniques (5 of 6) s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all 6.5 Using Cloned DNA Fragments to Study Gene Expression Te of., B rs fe ar, lar F s ve ro rc u 2_ la ni P a ec 0 U nt S ol.0 Blotting techniques identify specific DNA fragments in complex mixtures. e ta N M 02 Microarray and in situ hybridization techniques reveal mRNA expression, co- Th is a tic -34 regulation, and localization. As ub ry OL Recombinant DNA expression vectors enable regulated expression of exogenous B I o genes and production of proteins in prokaryotic and eukaryotic cells. k a h Eu S s r. D In situ hybridization can detect the activity of specific genes in whole and sectioned embryos s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo C) Drosophila at c M A) and B) embryo ity ss P C all Te of., B Mouse embryo probed for an rs fe ar, lar F probed for Sonic s mRNA ve ro rc u 2_ la hedgehog produced ni P a ec 0 mRNA U nt S ol.0 during e ta N M 02 trachea development Th is a tic -34 As ub ry OL In situ hybridization retains and reveals gene expression positional information in an organism. Assay steps: I B o Permeabilize cells in the specimen by treatment with detergent and a protease to expose localized mRNA to a DNA or RNA probe. a Hybridize a probe that is specific for the mRNA of interest and synthesized with nucleotide analogs containing k h Eu chemical groups that can be recognized by antibodies. Incubate the specimen in a solution containing an antibody that binds to the probe and is covalently linked to a S reporter enzyme (e.g., horseradish peroxidase (HRP) or alkaline phosphatase). s r. Provide substrate to the reporter enzyme to produce a colored precipitate where the probe has hybridized to the D ↳ beta target mRNA. galactase DNA microarray analysis can reveal differences in gene expression in fibroblasts under different experimental conditions DNA microarrays can be used to s tru , gy al n, c. evaluate the expression of D tio.S of or h.D ell -24 xa Ins MS iolo thousands of genes at one time. at c M A DNA microarray chip contains ity ss P C all Te of., B an organized array of thousands rs fe ar, lar F s of individual, closely packed, ve ro rc u 2_ la known gene-specific sequences ni P a ec 0 attached to the surface of a glass U nt S ol.0 microscope slide. e ta N M 02 Example: comparison of gene Th is a tic -34 expression in fibroblasts starved for serum and after serum As ub ry OL I addition using a DNA microarray B o a A green spot indicates higher expression in serum-starved cells. k h Eu A red spot indicates higher expression in serum(+) cells. A yellow spot indicates equal hybridization of green and red S s fluorescence, indicating no change in gene expression. r. D Some eukaryotic proteins can be produced in E. coli cells from plasmid vectors containing the lac promoter E. coli expression systems can produce large s tru , gy al n, c. quantities of proteins from a cloned gene/cDNA. D tio.S of or h.D ell -24 xa Ins MS iolo at c M (a) Expression vector plasmid: ity ss P C all Te of., B Contains the E. coli lac promoter and lacZ gene. rs fe ar, lar F s The lactose analog IPTG induces RNA ve ro rc u 2_ la polymerase to transcribe the lacZ gene, ni P a ec 0 U nt S ol.0 producing lacZ mRNA and the encoded β- e ta N M 02 galactosidase protein. Th is a tic -34 (b) Synthesize granulocyte colony-stimulating factor As ub ry OL I (G-CSF, a eukaryotic protein) in bacterial cells: Replace the plasmid lacZ gene with a cloned B o cDNA encoding G-CSF. Transform the plasmid into E. coli. k a h Add IPTG to induce transcription from the lac Eu promoter, producing G-CSF mRNA, which is S translated into G-CSF protein. s r. D G-CSF: granulocyte colony-stimulating factor IPTG: Isopropyl β-D-1-thiogalactopyranoside  β-Galactosidase Assay β-Galactosidase Assay/Miller’s Assay: Why to use LacZ/β-Gal as a reporter gene? s tru , gy al n, c. In the assay described above, the substrate o- D tio.S  In E. coli, the lacZ gene is the structural gene for β-galactosidase; of or h.D ell -24 xa Ins MS iolo nitrophenyl-β-D-galactopyraniside (ONPG) is at c M which is present as part of the inducible system lac operon. ity ss P C all used in place of lactose (in a buffer containing Te of., B  Protein product is stable and resistant to proteolytic degradation. sodium phosphate and magnesium chloride). rs fe ar, lar F s  Enzyme is easily assayed. ve ro rc u 2_ When the β-galactosidase cleaves ONPG, o- la  Easy and rapid method to assay β-gal activity in transfected cells. nitrophenol is released. ni P a ec 0 U nt S ol.0 This compound has a yellow color and absorbs  No expensive equipment. e ta N M 02 420 nm light. To measure β-galactosidase activity the Th is a tic -34 accumulation of yellow color (increase 420 nm As ub ry OL I absorbance)/minute is monitored. B o k a h Eu S Derived from the condensation of galactose and glucose, which form a β-1→4 glycosidic linkage s r. D https://www.promega.com/products/luciferase-assays/reporter-assays/beta_galactosidase- DOI: 10.1101/pdb.prot5423 enzyme-assay-system-with-reporter-lysis-buffer/?catNum=E2000  do get t Transient and stable transfection with specially designed plasmid vectors permits the expression of cloned genes in cultured animal cells s tru , gy al n, c. red Plasmid vectors: contain elements necessary for functioning in both E. coli and D tio.S of or h.D ell -24 xa Ins MS iolo animal cells: replication origin, selectable marker (e.g., ampr), and polylinker at c M (multiple cloning site) for insertion of a target sequence. ity ss P C all Te of., B rs fe ar, lar F s (a) Transient transfection: ve ro rc u 2_ la The plasmid vector contains a virus replication origin but is not faithfully ni P a ec 0 segregated into both daughter cells during cell division and is lost from U nt S ol.0 e ta N M 02 replicating cells over time. No selectable marker and media are used. Th is a tic -34 time The cDNA-encoded protein is produced for only limited a limited time. As ub ry OL (b) Stable transfection: I The plasmid vector carries an additional selectable marker such as neor, B o which confers resistance to G-418 (Geneticin, an analog of Neomycin sulphate and have similar mechanism as Neomycin). k a h Growth on G-418 medium selects the relatively few transfected animal Eu cells that integrate the exogenous DNA into their genomes. S Stably transfected cells continue to produce the cDNA-encoded protein s r. as as long as cell culture is maintained. long asthe theculture is maintained. D  Retroviral vectors can be used for the efficient integration of cloned genes into the mammalian genome, as part of Gene delivery strategy based awireit s tru , gy al n, c. centivirus A retrovirus is a type of virus that uses RNA as D tio.S of or h.D ell -24 xa Ins MS iolo its genetic material at c M ity ss P C all Lentivirus is capable of infecting both dividing Te of., B rs fe ar, lar F and postmitotic cells (e.g. neurons) and is s ve ro rc u 2_ la therefore widely used in neuroscience ni P a ec 0 experiments. Lentivirus is based on the human U nt S ol.0 immunodeficiency virus and has an 8-kb carrying e ta N M 02 capacity. Because the DNA integrates into the Th is a tic -34 genome, lentivirus delivery leads to long-term expression As ub ry OL I VSV-G (vesicular stomatitis virus) is an example B o of a viral coat protein that coats hybrid virus particles (formed via coinfection), making them a capable of infecting a wide variety of k h Eu mammalian target cell types, including hematopoietic stem cells, neurons, and muscle S and liver cells (https://doi.org/10.1016/B978-0-12- s r. 800511-8.00011-3) D Recombinant lentivirus particle production — three different plasmids are introduced into cells by transient transfection Lentivirus retroviruses increase the efficiency by which a modified gene can be stably integrated and expressed in animal cells. Recombinant lentivirus particle production — three different plasmids are introduced into cells by transient transfection: s tru , gy al n, c. First plasmid — vector plasmid: D tio.S of or h.D ell -24 xa Ins MS iolo Contains the cloned gene of interest next to a selectable marker such as neor flanked by lentivirus LTR (Long terminal at c M repeat) sequences. ity ss P C all Te of., B The left LTR sequence directs synthesis of an RNA molecule that carries lentiviral LTR sequences at either end. (It has rs fe ar, lar F s many properties of native retroviral RNA.) ve ro rc u 2_ la The LTR-bearing RNA can be packaged into viral particles and then introduced into a target cell by viral infection. ni P a ec 0 In the target cell, the LTR sequences direct copying the RNA into double-stranded DNA by reverse transcription and U nt S ol.0 integration of that DNA into chromosomal DNA. (one famous example is HIV) e ta N M 02 Second plasmid — packaging plasmid: Th is a tic -34 Carries the viral genes, except for the major viral coat protein, necessary for packaging LTR-containing viral RNA into a functional lentivirus particle. As ub ry OL Third plasmid — viral coat plasmid: I Encodes the expression of a viral coat protein (e.g., the vesicular stomatitis virus VSV-G protein) that coats hybrid B o virus particles, making them capable of infecting a wide variety of mammalian target cell types, including hematopoietic stem cells, neurons, and muscle and liver cells. k a Cell lentivirus particle infection: h Eu Engineered lentiviruses infect cells with such high efficiency that every cell in a population will receive at least one S copy of the lentivirus-borne plasmid. s The cloned gene flanked by the viral LTR sequences is reverse-transcribed into DNA, which is transported into the r. D nucleus. The cloned gene is integrated into the host genome, from where it is expressed.  2 types of mutations - chromosomal Molecular Genetic Techniques (6 of 6) Point mutation s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all 6.6 Altering the Function of Specific Genes by Design Te of., B rs fe ar, lar F s ve ro rc u 2_ Gene mutation can reveal clues to the function of its product. la ni P a ec 0 U nt S ol.0 Genes can be mutated by introducing point mutations or various transient e ta N M 02 or stable gene knockout techniques involving homologous recombination, Th is a tic -34 ↓ CRISPR-Cas9 gene editing, or interfering RNA. As ub ry OL Diffbtukort knockdown I in the B the sentence gene. o ) take ↳ out part of take out < The entire a sentence k h Eu in the gene S s r. D Homologous recombination with transfected disruption constructs can inactivate specific target genes in yeast ↳introducing a foreign object s tru , gy al n, c. Normal yeast genes can be replaced with mutant alleles by homologous D tio.S of or h.D ell -24 xa Ins MS iolo recombination. at c M ity ss P C all Te of., B (a) PCR primer construction for generating a disruption construct containing rs fe ar, lar F s a selectable marker gene: ve ro rc u 2_ la Two primers — each contains ~20 nucleotides (nt) homologous to both ↳ Acts For ni P a ec 0 as a selectable sequences flanking the target yeast gene connected to terminal X yeasta U nt S ol.0 coli marker sequences of a selectable marker gene such as the kanMX G-418- e. e ta N M 02 gene resistance gene (enables the selection of both kanamycin-resistant Th is a tic -34 transformants in Escherichia coli and G418-resistant transformants in the yeasts Saccharomyces cerevisiae). As ub ry OL I (b) Diploid Saccharomyces cells transformed with the disruption construct: B o Homologous recombination between the ends of the construct and the corresponding chromosomal sequences replaces the target sequence a with the marker gene. k h Eu Only recombinant diploid cells will grow on a medium containing G-418 (Geneticin antibiotic). S If the target gene is essential for viability, 50 percent of the haploid s r. D spores formed by recombinant diploid cell sporulation will be nonviable. https://www.youtube.com/watch?v=M_pUFziT3W4  Single-nucleotide mutations can be introduced into the genome using an s tru , gy engineered CRISPR-Cas9 system al n, c. in one Gopen reading frames DNA D tio.S of or h.D ell -24 xa Ins MS iolo M at c M Strand. ity ss P C all Te of., B rs fe ar, lar F s ve ro rc u 2_ la ni P a ec 0 U nt S ol.0 every species has a own e ta N M 02 species - specificpromoter ① Th is a tic -34 NLS: Nuclear Localization Signal As ub ry OL I B o generest k a h CRISPR (Clustered Regularly Interspaced Short Palindromic Eu Repeats) and CRISPR-associated (Cas) genes (9/12) S s r. D https://www.youtube.com/watch?v=4YKFw2KZA5o Gene editing: precise modification of a target gene by expression of the double-stranded DNA endonuclease Cas9 and a guide RNA (a) Protocol: Constructs: s tru , gy al n, c. Plasmid encoding Cas9 D tio.S Plasmid encoding the guide RNA — two parts: of or h.D ell -24 xa Ins MS iolo A sequence that folds into a hairpin scaffold structure that binds Cas9. at c M A sequence of ~20 nt corresponding to the targeted site in the gene. ity ss P C all Te of., B Expression of these components by transfection with plasmids or by direct injection of Cas9 mRNA and guide RNA. rs fe ar, lar F s (b) Cas9 mechanism: ve ro rc u 2_ la Base pairing between guide RNA and its complementary genomic DNA sequence directs the Cas9 complex to the targeted region of the ni P a ec 0 genome. U nt S ol.0 Two distinct Cas9 nuclease activities cleave both strands of the target DNA adjacent to the heteroduplex formed with the guide RNA. e ta N M 02 (c) Repair-target gene inactivation: two possibilities Th is a tic -34 Nonhomologous end joining (NHEJ) — repairs the double-stranded target gene cleavage, but removes a few bases at the cleavage site, which can inactivate gene function by producing a frameshift mutation. As ub ry OL Homology-directed repair (HDR) — Inclusion of a ∼100-nt single-stranded DNA segment that spans the sequences flanking the cleavage site along with Cas9 mRNA and the guide RNA causes homologous recombination repair, which can introduce single base changes in the B I repaired genomic DNA. o (d) Cleaved DNA repair: NHEJ: a Usually removes a few bases at the cleavage site. k h Eu Cleavage in a coding sequence — usually inactivates gene function by producing a frameshift mutation. (Knock-outs are possible) HDR: S Injection of ~100-nt single-stranded DNA segment that spans the cleavage along with Cas9 mRNA and the guide RNA — repairs s r. break with no base loss. D Single base changes can be introduced into the repaired genomic DNA. (Knock-ins are possible)  The loxP-Cre recombination system can knock out genes in specific cell types ↳ knock specific cell out types A loxP site is inserted in the introns on each s tru , gy al n, c. D tio.S side of a target gene essential exon by of or h.D ell -24 xa Ins MS iolo homologous recombination at c M ity ss P C all Te of., B A Cre mouse with one target gene knockout rs fe ar, lar F s ve ro rc u 2_ la allele and a cre gene from bacteriophage P1 linked to a cell-type-specific promoter ni P a ec 0 U nt S ol.0 incorporated into the mouse genome by non- e ta N M 02 homologous recombination (NHEJ) Th is a tic -34 (doi.org/10.1038/nrm.2017.48); doi.org/10.1146%2Fannurev-biochem-080320- As ub ry OL I 110356 B o loxP-Cre crossed mice produce Cre only in cells in a which the promoter is active — Cre catalyzes k h recombination to delete the essential exon in the Eu only functional allele in those cells S s r.  We got rid of Exon2 D https://www.youtube.com/watch?v=V70Uoi672-c  The loxP-Cre recombination system can knock out genes in specific cell types s tru , gy al n, c. Somatic cell recombination can inactivate target genes in specific tissues, at a specific D tio.S of or h.D ell -24 xa Ins MS iolo - development stage, or both. at c M Cre catalyzes recombination between loxP site-specific sites. ity ss P C all Te of., B ↓ Protocol: rs fe ar, lar F s ve ro rc u 2_ la A loxP site is inserted in the introns on each side of a target gene essential exon by ni P a ec 0 homologous recombination, producing a loxP mouse. U nt S ol.0 (loxP sites are in introns and do not disrupt target gene function.) e ta N M 02 Th is a tic -34 A Cre mouse with one target gene knockout allele and a cre gene from bacteriophage P1 As ub ry OL linked to a cell-type-specific promoter incorporated into the mouse genome by I nonhomologous recombination. (The cre insertion does not affect other gene function.) B o a loxP-Cre crossed mice produce Cre only in cells in which the promoter is active — Cre k h Eu catalyzes recombination to delete the essential exon in the only functional allele in those S cells. s Different cell-specific promoters linked to cre cause gene knockout in other specific cell types. r. D  Processing of mRNA Drosha/Dicer, miRNA transcription and processing: = questions 8th ed, ch 10 - ↳ micro RNN 9th ed ch 9: post txn gene control RNA polymerase II transcribes primary miRNA transcripts (pri-miRNA) – folds to s tru , gy al n, c. form double strand region D tio.S of or h.D ell -24 xa Ins MS iolo Nuclear double-strand RNA–specific endoribonuclease Drosha and double-strand at c M t RNA–binding protein DGCR8 (Pasha in Drosophila) bind pri-miRNA double-strand ity ss P C all Te of., B regions ↳ 8 chromosomes rs fe ar, lar F s Drosha cleaves the pri-miRNA – generates a ~70-nucleotide pre-miRNA ve ro rc u 2_ la '. ' ↑ Exportin 5 – nuclear transporter transports processed pri-miRNA to the cytoplasm ni P a ec 0 U nt S ol.0 Dicer (dsRNA sp. RNase) in conjunction with the double-stranded RNA–binding e ta N M 02 protein TRBP (Loquacious in Drosophila) – processes pre-miRNA into a double- Th is a tic -34 stranded miRNA with a two-base single-stranded 3′ end (on both strands) NESZ nuclear exposed As ub ry OL RISC complex (RNA Induced Silencing Complex)– binds one of the two strands. I signal B o incorporates mature miRNA into complex with Argonaute proteins mRNA translation inhibition: a miRNA-RISC complexes associate with target mRNPs (nuclear messenger k h Eu ribonucleoprotein) by base pairing between the Argonaute-bound mature miRNA S and complementary regions in the 3′ UTRs of target mRNAs s The more RISC complexes bound to the 3′ UTR of an mRNA, the greater the r. D repression of translation https://www.youtube.com/watch?v=t5jroSCBBwk