Lecture 2- Transformation techniques.pdf

LECTURE 2 TRANSFORMATION BIOC 3260 PRINCIPLES OF BIOTECHNOLOGY DR. ANGELA T. ALLEYNE 1  1. Explain the difference between gene transfer via transformation and transfection...

LECTURE 2 TRANSFORMATION BIOC 3260 PRINCIPLES OF BIOTECHNOLOGY DR. ANGELA T. ALLEYNE 1  1. Explain the difference between gene transfer via transformation and transfection 2. Draw and label simple gene construct diagrams and vectors 3. Differentiate between cloning and expression vectors LEARNING 4. Differentiate between biological, chemical and OUTCOMES physical gene/nucleotide transfer methods 5. Describe electroporation and biolistic (gene-gun) delivery At the end of this lecture 6. Discuss the analysis of gene transfers you will be able to: 7. Describe general preparation of gene libraries 8. Explain the merits of RNA transfer 2 A model organism is a non-human species , whose entire genome is known and scientists the lab to investigate , use them in and understand biological processes. MODEL ORGANISMS 3  First observed naturally by Griffith ( 1928) when pathogenic and non- pathogenic bacterial strains of pneumococcus bacteria were mixed together Avery, Macleod and McCarty later GENE demonstrated that the principle molecule responsible for changing or TRANSFER transforming the cells was DNA- they termed the method genetic Transfer of a gene from transformation one DNA molecule to Bacteria can be transformed naturally another DNA molecule ( Results may be transient or stable not necessarily to a new 4 transfection organism).  TRANSGENES AND GENE CONSTRUCTS An organism that gains new genetic information from the addition of foreign DNA is described as transgenic, while the introduced DNA is called the transgene This Photo by Unknown Author is licensed under CC BY-SA Gene construct refers to any recombinant DNA molecule that has been assembled by genetic engineering. 5  GENERAL PROPERTIES OF CLONING VECTORS Small size, making them easy to manipulate once they are isolated Bacteria plasmids Easy to transfer from cell to cell (usually by transformation) Easy to isolate from the host organism A polylinker is a segment short of DHD which contains up to Easy to detect and select ~ 20 restriction sites. Multiple copies help in obtaining large amounts of DNA facilitates insertion of foreign DND without disrupting the rest of the plasmid Clustered restriction sites (polylinker) to allow 6 insertion of cloned DNA Clark Biotechnology 2nd edition  INSERTIONAL INACTIVATION Transformed cells or cells with new gene constructs become sensitive to a second antibiotic. Cells without 7 an insert remain resistant to the antibiotic β -galactosidase is expressed as two protein fragments, which when assembled forms a functional protein. 8  ALPHA COMPLEMENTATION Blue colonies- cells without an insert in the White colonies- cells with an insert and the plasmid and β -galactosidase is active. X-gal is alpha fragment is not made and β - split to form a blue dye. 9 galactosidase is inactive. X-gal is not split. 10 SHUTTLE VECTORS Contains origins of replication (ORI) for two different organisms/hosts plus any other sequences necessary to survive in either organism. The inserted DNA can be manipulated in two different cell types. Yeast shuttle vectors that are based on the two plasmids have the components needed for survival in yeast and bacteria, plus antibiotic resistance and a polylinker. 11 SHUTTLE VECTORS The E. Coli component of a yeast shuttle vector includes an origin of replication and a selectable marker, such as antibiotic resistance like beta-lactamase. The yeast component of a yeast shuttle vector includes an autonomously replicating sequence (ars), a yeast centromere (cen), and a yeast selectable marker. 7.14D: SHUTTLE VECTORS AND EXPRESSION VECTORS - BIOLOGY LIBRETEXTS 12  EXPRESSION VECTORS Expression vectors must have Expression vectors are used for techniques expression signals such as a: such as site-directed mutagenesis 1. strong promoter, Cloning vectors, similar to expression 2. strong termination codon, vectors, but the plasmid is then added into bacteria for replication purposes. 3. distance between the promoter and the cloned DNA vectors that are used in many gene, molecular-biology gene-cloning experiments need not result in the 4. transcription termination expression of a protein. sequence, 5. ptis (portable translation 13 initiation sequence or tir).  a multiple cloning site, a promoter, a repressor, and a selectable marker. This Photo by Unknown Author is licensed under CC BY-SA 14 CONSTRUCT YOUR OWN PLASMID 15  E. coli transformation TRANSFORMATION A genetic change brought about by picking up naked strands of DNA and expressing it Usually associated with prokaryotic cells. Taken from: http://images.slideplayer.com/24/7020713/slides/slide_6.jpg 16  TRANSFECTION The introduction of foreign nucleic acid material into eukaryotic cells. May involve opening transient pores in the cell plasma membrane, to allow uptake of material Methods include Electroporation Gene gun Lasers Sonogram etc. 17 http://www.nscientific.com/wp-content/uploads/2013/08/What-is-transfection-1024x735.jpg  In transient transfection, the transfected DNA or RNA is expressed in target cells, but the nucleic acids are not integrated into the host cell genome. Nucleic acids may be transfected in the form of a plasmid Transiently transfected genetic materials may be lost by environmental factors and cell division. Usually results in high expression levels that TRANSIENT persist for a short period. TRANSFECTION RNA - for 24-72 hours following transfection, 18 DNA for 48-96 hours following transfection.  Stable transfection, long-term expression of a transgene by integrating foreign DNA into the host nuclear genome or A selection method is used to isolate stably transfected cells. Stable transfection is useful in long-term genetic and pharmacology studies in which STABLE large-scale protein production is needed. TRANSFECTION 19  Short lived expression Taken from 20 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911531/pdf/216_2010_Ar ticle_3821.pdf  THREE METHODS: 1. HORIZONTAL GENE TRANSFER 2. TRANSDUCTION- VIRUS-MEDIATED BIOLOGICAL 3. CONJUGATION- FUNGI METHODS 21  TRANSDUCTION BIOLOGICAL A viral vector to carry a specific nucleic acid sequence into a host cell. Retroviruses, such as lentiviruses are often used for stable transfection. Adenovirus, adeno-associated virus (AAV) and herpes virus do not guarantee stable transfection. Integrase - enzyme produced by viruses such as retroviruses that facilitate the integration of foreign genetic materials into the host genome 22  CHEMICAL TRANSFECTION- TWO METHODS The cell membrane sold can in on itself Micells are formed facilitating the movement of foreign DMD into cells. Liposomal-based transfection reagent is a chemical that enables the , formation of positively charged lipid aggregates that could merge smoothly with the phospholipid bilayer of the host cell to allow the entry of the foreign genetic materials with minimal resistance Non- Liposomal: chemicals other than liposomes are used, including calcium phosphate, dendrimers, polymers, nanoparticles and non-liposomal lipids 23  CHEMICAL METHODS DNA is taken up from the surroundings via the cell Some chemicals used include: membrane. Calcium phosphate, Calcium The DNA forms a chemical complex with an chloride, overall positive charge, allowing it to Cationic Lipid interact with the negatively charged cell membrane and promote uptake by Other polymers - poly-L-lysine endocytosis (PLL), Polyethylene glycol ( PEG), Polyethylenimine (PEI) A lipophilic complex contains the DNA polyphosphoester, etc. which then fuses with the cell membrane and deposits the transgene directly into the Dendrimers – branched 24 cytoplasm. molecules that complex with DNA CALCIUM PHOSPHATE TECHNIQUE Binding positively charged calcium ions (ca2+) with the negatively charged nucleic acids to form a precipitate before being taken up by host cell. Since the cells must be coated by the calcium complex, monolayers of cells must be used for maximum efficiency. Gives low efficiency (approx. Only 1- 2% transfection) Dependent on conc of the reagents and cells 25  LIPOSOMES Cationic lipids form liposomes are micellar structures developed for gene called liposomes delivery Cationic polymers, such as polylysine, polyamidoamine and Polyethylenimine (PEI) polyethyleneimines has been shown to (PEI) interact electro- mediate effective in statically with DNA to vitro and in vivo gene form compact transfer. structures called lipoplexes 26  27 https://www.researchgate.net/profile/Alan_Parker5/publication/9035177/figure/fig1/AS:340670498787335@1458233584979/Figure-1-Lipoplex-mediated- transfection-and-endocytosis-Cationic-lipids-forming.png  DNA is deposited directly into the cell by using a physical force. This includes: 1. Electroporation (chemical and physical method) 2. Sonoporation 3. Micro-injection 4. Scrape loading 5. Particle bombardment 6. Lasers- a laser creates a temporary photopore on the cell membrane PHYSICAL which DNA can pass through. Usually METHODS operates on one cell at a time. 28  A mechanical method used for the introduction of polar molecules into a host cell through the cell membrane. Widely used for the introduction of transgenes into bacterial, fungal, plant and animal cells. ELECTROPORATION It involves use of a large electric pulse (1-1.5 kV) Used for difficult cells-, that temporarily disturbs the phospholipid bilayer, stem cells and B cell allowing the passage of molecules such as DNA lines ? pros + cans high voltage may cause cell necrosis, apoptosis, and permanent cell damage DNA. 29  The basis of electroporation is the relatively weak hydrophobic/hydrophilic interaction of the phospholipids bilayer and ability to spontaneously reassemble after disturbance. https://bio.libretexts.org/@api/deki/files/8783/OSC_Microbio_12_01_electropor.jpg?revision=1&size=bestfit&width =1200&height=427 30  TYPICAL PROTOCOL FOR ELECTROPORATION 1. Inoculate a colony into ~50 ml (no salt) LB and grow at 37°C overnight. 2. Add ~25 ml culture medium into 1 l lb medium. 3. Grow the cells at 37°c in a shaking incubator. 4. Grow cells to an a600 of ~0.6-0.7. This represents the bacteria’s log-phase growth. Cells in this phase are growing rapidly, and are healthy and uniform. 5. Pour ~250 ml into a tube and spin down in a centrifuge at 4°c. 6. Remove supernatant and resuspend cells in dh2o. 7. Repeat centrifugation/removal of supernatant several times. 31 8. Resuspend in 10% glycerol and keep in freezer until ready to use or keep on ice  ELECTROPORATION PROTOCOL ( CONT’D) 9. Prepare the DNA you want to put into the cells (i.E. Dilute it). 10. Pipette some (~100 µl) cells and dna (~1 µl) into a cuvette.* 11. After making sure the settings on the electroporator are correct, put the cuvette in and press the button. Your settings should maximize the number of transformed bacteria while also keeping as many alive as possible. 12. Within 30 seconds of electroporation, pipette about half a ml of soc (recovery medium). SOC is buffer comprised of salts, glucose, amino acids (tryptone) and some yeast extract. Mix. 13. Let the cells recover at 37°c in a shaking incubator for about an hour. 32 14. Plate the cells and let them grow.  Typical scheme cuvette Electroporator Taken from: 33 http://blog.universalmedicalinc.com/wp-content/uploads/sites/136/gallery/postimages/electroporation- diagram.png  MICROINJECTION ̶ Direct needle transfer ̶ DNA is inserted directly into nucleus ̶ Versatile technique for introducing exogenous material into cells, or transferring cellular components between cells ̶ Common clinical applications include in vitro fertilisation (IVF), and gene therapy ̶ Used to introduce DNA constructs, antibodies, short interfering RNAs (siRNAs), and peptides. 34  Easier to dow bacterial cells Taken from Clark Biotechnology: file:///D:/Sem%20I- %20Teaching/BIOC%203260- %20PO%20Biotech/Refs/biotechnology%20clark.p df Scrape loading- the cells are scraped off the bottom of the culture dish, the membranes break open, allowing the oligonucleotides to enter. 35 Oligonucleotides can be tagged with a fluorescent label  PARTICLE BOMBARDMENT Gene gun DEVELOPED AT CORNELL UNIVERSITY IN 1987. THEY INTRODUCED CAT (CHLORAMPHENICOL ACETYL TRANSFERASE) GENE INTO ONION EPIDERMAL CELLS AND DETECTED TRANSIENT GENE ACTIVITY. SHOOTS FOREIGN DNA INTO PLANT CELLS OR TISSUE AT A VERY HIGH SPEED. USES COMPRESSED GAS TO DELIVER DNA-COATED HEAVY METAL PARTICLES. GOLD BEADS ARE PREFERABLE BECAUSE TUNGSTEN CAN BE TOXIC TO SOME PLANTS. THE BEADS ARE PLACED AT THE END OF A PLASTIC BULLET. ALSO KNOWN AS PARTICLE GUN METHOD, BIOLISTIC PROCESS, MICROPROJECTILE BOMBARDMENT OR PARTICLE ACCELERATION. 36  ̶ DNA-coated microscopic particles are accelerated at high speed by helium gas within a vacuum and penetrate target cells. ̶ Useful for tissues rather than cells ̶ successfully used to generate transgenic wheat plant https://www.intechopen.com/source/html/30876/media/image1.png 37 38  SEVERAL METHODS : ̶ MICROSCOPY ̶ CELL COUNTING ANALYSIS OF TRANSFECTION ̶ REAL TIME PCR ̶ WESTERN BLOTTING ̶ FLUORIMETRY ETC. 39  Measured by use of reporter systems Green Fluorescent Protein (GFP)— TRANSFECTION visualized directly by fluorescence microscopy EFFICIENCY Quantitative measurement by Flow cytometry, or by PCR Luciferase The luciferase assay allows for a quantitative readout of photon emission 40  β-galactosidase (β- gal) colorimetric qualitative of TRANSFECTION quantitate assays EFFICIENCY Can be calculated by measuring transformation efficiency : # colonies on plate/ng of DNA plated X 1000 ng/µg 41  LUC+ tobacco First reporter gene: CAT: chloramphenicol acetyl transferase (chromatography and autoradiography) nos or ocs: nopaline or octopine synthase (chromatography) uidA or gusA (GUS): MUG or X-gluc (color or fluorescence) Jefferson (1987) luc (firefly luciferase) luciferin (bioluminiscence) Ow et al. (1986) gfp (green fluorescent protein) no substrate, UV irradiation. negative positive positive GFPpositive expression in rice callus positive positive  COMPARISON OF METHODS- CHEMICAL transfection efficiency of chemical methods is largely dependent on factors such as CONS nucleic acid/chemical ratio, solution pH, and cell membrane conditions. results in low transfection efficiency, especially in vivo, compared with virus- mediated methods. relatively low cytotoxicity, no mutagenesis, Pros no extra-carrying DNA, and no size limitation on the packaged nucleic acid. Chemical transfection efficiency also varies depending on cell type. 43  Microinjection demands skill, often causes cell death, and is very labour-intensive unreliable, tricky, low (or no) success rate ( when not well done). CONS Biolistic methods requires expensive instruments and causes physical damage to samples. Laser techniques require an expensive laser-microscope system. PHYSICAL 44  PHYSICAL Biolistics is straightforward and reliable. Electroporation is easy and rapid; it is able to transfect a large number of cells in a short time once optimum electroporation conditions are Pros determined. Laser method enables one to observe the transfecting cell and to make pores at any location on the cell. This method can be applied to very small cells, because it uses a laser. 45 Traditionally, RNA transfection is less widely However it is widely used today in RNAi used than DNA because ( RNA interference of the instability of the or gene silencing) RNA RNA TRANSFER If using mRNA, the features such as a 5' cap, internal ribosomal entry site, or poly-A tail can have a significant effect on efficiency of transfection RNA is usually transiently transfected 46 RNA has several advantages over plasmid or viral approaches. The protein product is produced rapidly after the transfection. Plasmid based DNA approaches require a strong promoter drives the expression of the gene of interest in cells. Typically produces an extremely high rate of transfection. One can introduce any number of mRNAs into a cell, thereby overcoming overexpression of the genes. 47  RNA has several advantages over plasmid or viral approaches. Various forms of RNA, such as mRNA, in vitro transcribed RNA, viral RNA, RNA oligos, siRNA, and ribozymes, can be used for transfection There is no risk of integration into the host genome, cell cycle- independent transfection efficiency. There is no need for immune inducible vectors ( Ab resistant), and adjustable and rapid expression. 48  PDF RESOURCES https://www.Ncbi.Nlm.Nih.Gov/pmc/articles/PMC2911531/p df/216_2010_article_3821.Pdf https://www.Ncbi.Nlm.Nih.Gov/pmc/articles/PMC2849731/p df/nihms179396.Pdf https://translational- medicine.Biomedcentral.Com/track/pdf/10.1186/1479-5876- 2-39?Site=translational-medicine.Biomedcentral.Com Transfection types, methods and strategies: a technical review [peerj] 49