Genetically Modified Cells and Organisms PDF

Genetically Modified Cells and Organisms Andreas Ivessa, Ph.D. Department of Cell Biology and Molecular Medicine [email protected] April 10, 2024 Figures from “Molecular Cell Biology” (6th edition) Objectives: What is the difference between correlation and causation? How is a specific gene i...

Genetically Modified Cells and Organisms Andreas Ivessa, Ph.D. Department of Cell Biology and Molecular Medicine [email protected] April 10, 2024 Figures from “Molecular Cell Biology” (6th edition) Objectives: What is the difference between correlation and causation? How is a specific gene identified (overall schema)? What is the difference between phenotype and genotype? What are recessive and dominant mutations? What is the difference between mitosis and meiosis? What are temperature sensitive mutants? What is suppression and synthetic lethality? How does DNA cloning work? What does a restriction enzyme recognize in DNA? Which genes and functional elements does a plasmid need to contain to be propagated in bacteria? How does DNA ligation work? How does the polymerase chain reaction (PCR) work? Explain the principles of the DNA gel electrophoresis. What is a cDNA and genomic DNA library? What is the function of the reverse transcriptase? What is the principle of DNA sequencing? How is a gene deleted in baker’s yeast? How is a gene knocked-out in the entire mouse and it deleted in a specific tissue? How does CRISPR-Cas genome engineering work? How is the expression of a gene inactivated by the RNA interference technique? How is trans-gene expression carried out in mammalian cell culture? How can gene expression be monitored? Correlation -- Causation Amount of protein A increases during aging young Correlation old aging The increase in the amount of protein A during aging may just be a coincidence and may have no impact on aging There is a need to change the amount of proteins (deletion of genes, over-expression of proteins) to distinguish between correlative and causative relationships. Young cells lacking protein A Shorter lifespan – Cells age faster or Longer lifespan – Cells live longer or NO change in lifespan Causation The lack of protein A has an impact on lifespan The increase in the amount of protein A correlates with aging 1. Genetic analysis of mutations 2. DNA cloning and characterization 3. Knockout and transgenic Forward and Reverse Genetics Genes are not altered, no defects to observe Gene(s) is/are missing/altered, has/have defect(s) DNA mRNA protein GENOTYPE --- PHENOTYPE Split into two parts Identical twins: -- Identical genotype Meaning: Identical DNA -- Different phenotype Meaning: Differences in gene expression, protein modification, protein activity, … (called: epigenetics) GENOTYPE --- PHENOTYPE Examples for changes in the genotype Gene has alterations in its sequence Part of the gene is missing / duplicated Examples for changes in the phenotype Cells with mutations are growing slowly Animals with a disease have a shorter lifespan Cells / animals with mutations have a longer lifespan, are better susceptible to external factors (e.g., heat stress) (Enhancer) Gene-regulatory sequences bind transcription factors Mutations Encodes the protein Promoter Open Reading Frame Chromosomal Mutation Mutation DNA (Point-mutations, deletions rearrangements) Expression level changed Proteins: Expression level Enzyme activity Protein-protein interactions Protein-DNA/RNA interactions Protein-membrane interactions Protein localization RNA: Micro RNAs Dominant and Recessive Mutant “Gain of Function” and “Loss of Function” Mutant Although a functional wild type gene is present, the mutant gene “dominates” the phenotype. The phenotype caused by the mutant gene is rescued by the presence of the functional wild type gene. Common Inherited Human Diseases Huntington's disease: Defective neural protein (huntingtin) may assemble into aggregates causing damage to neural tissue. Cystic fibrosis: Defective chloride channel (CFTR) in epithelial cells leads to excessive mucus in lungs. DOMINANT RECESSIVE Figure 5-35 Mitosis -- Meiosis Mitosis (normal cell division): homologous chromosomes 2N 4N Meiosis (creation of germ line cells, e.g. oocytes/sperm): 2 cell divisions X X pairing and cross-overs 2N 4N (exchange of genetic material between homologous chromosomes) diploid meiosis haploid MUTANT PHENOTYPE diploid meiosis haploid diploid diploid meiosis haploid WILD TYPE PHENOTYPE diploid meiosis haploid diploid Essential and non-essential genes NO Parallel pathway Parallel pathway A Gene 1 Gene X1 X B Gene 2 C Gene X2 A Gene 1 B Gene 2 C One pathway: Multiple pathways: Non-viable: Viable: Gene 1 is essential Gene 1 is non-essential Conditional mutations can be used to study essential genes in yeast Mutagen: Changes the genetic information of DNA...A T T G A G......T A A C T C... Mutagen...A T T G A G......T A T C T C... Replication...A T T G A G......T A A C T C... Codon usage...A T A G A G... altered...T A T C T C... Temperature-sensitive mutants: Normal growth and function of the studied protein at low temperature (i.e. 23°C) Growth arrest and impaired function of the studied protein at high temperature (i.e. 36°C) (e.g. protein is produced but rapidly degraded at high temperature) Figure 5-6 normal growth growth arrest / impaired function Temperature-sensitive mutants Normal growth and function of the studied protein at low temperature (i.e. 23°C) Growth arrest and impaired function of the studied protein at high temperature (i.e. 36°C) (e.g. protein is produced but rapidly degraded at high temperature) * * messenger RNA Gene mutated 23°C 36°C Protein Properly folded Un-folded Proper function Protein targeted to degradation Conditional mutations can be used to study essential genes in yeast Is the same gene or are different genes mutated in these mutants? Obtain likely numerous mutants that can grow at 23°C, but not at 36°C. normal growth Figure 5-6 growth arrest / impaired function Genetic complementation analysis determines whether recessive mutations are in the same or in different genes Haploid yeast strains can easily be converted to their opposite mating type. a to alpha alpha to a cdc mutants in yeast: Cell division cycle genes. Mutations in those genes cause cells to stop dividing in the cell cycle (G1, S, G2, M). Figure 5-7 Crossing of mutant strains 1 2 3 4 5 1 1 1 1 2 1 3 1 4 1 5 2 2 1 2 2 2 3 2 4 2 5 3 3 1 3 2 3 3 3 4 3 5 4 4 1 4 2 4 3 4 4 4 5 5 5 1 5 2 5 3 5 4 5 5 Mating type alpha Mating type a Genetic complementation analysis determines whether recessive mutations are in the same or in different genes Haploid yeast strains can easily be converted to their opposite mating type. a to alpha alpha to a cdc mutants in yeast: Cell division cycle genes. Mutations in those genes cause cells to stop dividing in the cell cycle (G1, S, G2, M). Figure 5-7 Analysis of double mutants often can order the steps in biosynthetic pathways Figure 5-8 Mutations that result in genetic suppression or synthetic lethality reveal interacting or redundant proteins Figure 5-9 1. Genetic analysis of mutations 2. DNA cloning and characterization 3. Knockout and transgenic DNA Cloning DNA fragment + Vector (Plasmid) Recombinant DNA Replication of recombinant DNA within host cells Isolation, sequencing, and manipulation of purified DNA fragment A plasmid (vector) contains sequences to maintain it in the host (e.g. replication origin, selection marker gene) Cleavage of palindromic sequence by restriction enzyme palindromic Daniel Nathans, Werner Arber, and Hamilton O. Smith Nobel Prize 1978 5’ 5’ EcoRI: enzyme derived from the bacterium Escherichia coli which cuts foreign DNA. Restriction enzymes are defense mechanisms against invading viruses in bacteria. Bacterial host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Figure 5-11 Restriction Enzymes and Their Recognition Sequences ENZYME SOURCE MICROORGANISM RECOGNITION SITE ENDS PRODUCED BamHI Bacillus amyloliquefaciens 5’ Sticky Sau3A Staphylococcus aureus 5’ Sticky NotI Nocardia otitidis-caviarum 5’ Sticky SmaI Serratia marcescens Blunt KpnI Klebsiella pneumonia -G-G-T-A-C-C-C-C-A-T-G-G- 3’ Sticky Frequency of recognition sequences: 44 (256bp), 46 (4,096bp), 48 (65,536bp) DNA ligation and multi-cloning sites Replication origin gap T4 DNA ligase Multi-cloning site Marker gene to select cells containing the plasmid Figure 5-12 Figure 5-13 1 94°C 55°C Polymerase Chain Reaction (PCR) The used DNA polymerase (called Taq polymerase) is 94°C heat resistant. 72°C 2 55°C 4 Taq polymerase derives from the thermophilic 72°C bacterium Thermus aquaticus which lives in hot springs. 94°C 55°C 72°C 8 Kary Mullis Nobel Prize 1993 Taq polymerase was found by Tom Brock (1965) Figure 5-23 Figure 5-24 DNA cloning in a plasmid vector permits amplification of a DNA fragment Cells with the plasmid express the protein ampR that confers resistance to the antibiotic ampicillin. Figure 5-14 DNA electrophoresis: Separation of DNA molecules by agarose gel electrophoresis technique according to molecular weight. DNA is negative: DNA migrates therefore to the positive electrode. Figure 5-19 DNA is visualized in the presence of UV light by a dye that incorporates (intercalates) into DNA. DNA is charged negatively on the surface DNA electrophoresis: Separation of DNA molecules by agarose gel electrophoresis technique according to molecular weight. DNA is negative: DNA migrates therefore to the positive electrode. Figure 5-19 DNA is visualized in the presence of UV light by a dye that incorporates (intercalates) into DNA. Cloning of a gene by complementation of a mutant gene in temperature sensitive mutant cells DNA genes A * B C D Mutation in gene C DNA A B FUNCTIONAL gene C C * C Mutation in gene C Replication origin Selection marker D plasmid Grows at 23°C DOES NOT grow at 37°C Grows at 23°C GROWS at 37°C Genomic versus cDNA library Library: collection of various DNA fragments Genomic DNA library DNA mRNA DNA mRNA DNA RNA genes A B C D Complementary DNA library (cDNA library) Tissue A : (e.g. brain) Genes A and D expressed Tissue B : (e.g.: heart) genes A B C D Genes A and B expressed Tissue A and B have … same chromosomal DNA, but have different genes expressed Therefore: Same genomic libraries – Different cDNA libraries Construction of a cDNA library (complementary) The cDNA library reflects the transcription profile of cells or tissues under specific conditions. Reverse Transcriptase RNA Reverse Transcriptase RNA/DNA DNA Figure 5-15 Construction of a genomic DNA library (bacteria) (yeast) (bacteria) (yeast) (yeast) A genomic DNA library represents the information of the entire nuclear genome. overlapping DNA fragments Screening of a genomic library Transform plasmid DNA into yeast cells (cdc yeast mutants show impaired cell cycle progressions at the nonpermissive temperature) Screening of a genomic library Plasmid with URA3 (maintaining plasmid) and genomic DNA fragments URA3 functional A mutant Genomic DNA A temperature sensitive mutation in gene A : Cells grow at 23°C do not grow at 37°C (having no plasmid with gene A) URA3 URA3 B C NO Complementation Complementation B C URA3 gene is needed to maintain plasmid in yeast cells A B C NO Complementation A B C Genes B and C are unrelated to A Growth 23°C 37°C + + + - + - Determination of the sequence of DNA DNA polymerases synthesize DNA from 5’ into 3’ direction. Sequencing of DNA Sequencing of DNA Conventional sequencing gel (polyacrylamide): Sequencing of DNA using nucleotides labeled with 4 different fluorophores G A T C WholeGenomeSequencing WholeExomeSequencing Break up DNA into short fragments (~350 bp) TranscriptomeSequencing mRNA with poly A tail After selecting mRNA from total RNA pool Repair ends: add adaptors Synthesize first and second strand Break up RNA and perform random priming Repair ends: add adaptors Capture fragments Containing exons Wash uncaptured DNA Sequence Sequence Alignment Final data is ~10 GB Alignment Final data is ~150 GB Alignment Final data is ~10 GB Whole-Genome-Sequencing 1. Genetic analysis of mutations 2. DNA cloning and characterization 3. Knockout and transgenic Knockout (null mutant) of specific genes in yeast: Targeted mutagenesis by homologous recombination (directs the PCR fragment to the gene which should be deleted) kanMX: Kanamycin (G418) resistance gene Figure 5-39 Knockout (null mutant) of specific genes in yeast: Targeted mutagenesis by homologous recombination kanMX Search for homology Example:...CTAGGTA... antibiotic...GATCCAT... (Meiosis) Figure 5-39 Targeted mutagenesis in mouse by homologous recombination ES cells: Embryonic Stem cells tkHSV: Thymidine-kinase converts ganciclovir into a toxic nucleotide ES cells: Embryonic Stem cells Means: knock-out construct has inserted at the site of interest or somewhere else in the nuclear genome. Resistant to ganciclovir: knock-out construct has likely inserted at the site of interest. Sensitive to ganciclovir: knock-out construct has likely inserted somewhere else in the nuclear genome. Knockout mouse Gene of interest is knocked out in ALL tissues of the mouse ES cells: Embryonic Stem cells Figure 5-41 Knockout in specific cells by loxP - Cre recombination e.g., only active in heart cells (recombinase which recognizes loxP sites) Figure 5-42 Modifying DNA by using the CRISPR-Cas system: Clustered regularly-interspaced short palindromic repeats (CRISPR) Was first observed in bacteria to recognize and digest foreign DNA Bacteriophage (like a virus) CAS CRISPR-associated proteins e.g. nuclease Cas9 to digest DNA Foreign DNA Digestion to small pieces (foreign DNA) Pieces of foreign DNA are stored in the bacterial genome (DNA) as a memory to protect cells from further attack RNA (CRISP RNA) Modifying DNA by using the CRISPR-Cas system: Clustered regularly-interspaced short palindromic repeats (CRISPR) Nuclease Cas9 Chromosomal DNA Base-pairing RNA/DNA (specific gene) sgRNA (Single-guide RNA) to recognize specific DNA region Double strand break Repair of the break A few applications of the CRISPR-Cas9 system: Genome editing of multiple genes in one step (insertions/deletions, precise change) Gene regulation (transcriptional activation/repression) Future directions (human therapeutics, ecological engineering) The CRISPR-Cas9 system changes the information on DNA level Modifying DNA by using the CRISPR-Cas system: Clustered regularly-interspaced short palindromic repeats (CRISPR) Nuclease Cas9 crRNA (CRISPR RNA) tracrRNA (trans-activating CRISPR RNA) Assembly of the Nuclease and the two RNAs in the nucleus by nuclease Cas9 PAM (protospacer adjacent motifs): (gene of interest) Are important to guide crRNA and tracrRNA to the gene of interest NHEJ: Non-homologous- HDR: Homology Directed Repair end-joining One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering Multiple Gene targeting in embryonic stem cells Wang, …, Jaenisch, 2013 May, Cell 9;153(4):910-8 5 different genes to be modified Embryonic stem cells One Step Generation of Mice With Multiple Mutations NHEJ: Non-homologousend-joining HDR: Homology Directed Repair Inactivation of genes by RNA interference (RNAi) using siRNA (small interfering RNA) : The RNA expressed by the gene of interest is only degraded. The gene itself on chromosomal DNA stays intact. RISC (RNA-induced silencing complex) Dicer is an RNase endonuclease which generates dsRNA fragments 1. Genetic analysis of mutations 2. DNA cloning and characterization 3. Knockout and transgenic Mammalian cell culture: Transgenic (ectopic expression) only in those cells which were transfected with the plasmid Mammalian cell culture: Transgenic (ectopic expression) Mammalian cell culture: mRNA molecules packed in lipid nanoparticles and delivered to target cells: These are taken into the cells by endocytosis. The mRNA is released and translated. This method is used to produce the spike protein of the SARSCoV-2 virus. Future applications are to cure diseases including cancer. MHC: major histocompatibility complex Transgene expression Northern analysis: -- Purify RNA from cells or tissues -- Separate RNAs by agarose gel electrophoresis -- Transfer RNAs to membrane -- Hybridize to specific probe (e.g., radioactive labeled DNA) RNA in situ hybridization: probe cells or tissues with fluorescently labeled single stranded DNA Figure 5-27 Figure 5-28 Determining the transcriptional activity genome-wide using microarrays RNA Reverse Transcriptase RNA/DNA DNA Science. 1995 Oct 20;270(5235):467-70. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Schena M, Shalon D, Davis RW, Brown PO. Determining the transcriptional activity genome-wide using microarray Sample question: The DNA ligase enzyme A. cuts DNA at specific sites B. connects physically two DNA fragments C. converts restriction DNA fragments with sticky ends to blunt ends D. synthesizes DNA E. synthesizes DNA using an RNA template