Course Contents DNA and RNA isolation Quantification of DNA and RNA Primer designing PCR

1. Course Contents • DNA and RNA isolation • Quantification of DNA and RNA • Primer designing • PCR • Electrophoresis • Sequencing • Karyotyping • Restriction Mapping • Flow cytometry • Hybridization • Western blotting • Southern blotting • Northern blotting • FISH

2. Tissue culturing • Slide Preparation and Cell Stains • Agar plate preparation and streaking for the purpose of individual colony isolation • Bacterial Growth on selective agar • Quantification: Colony Forming Units (CFU) • Dilution Plating • Identification and characteristics of colonies • Transfection • Transduction • Transformation • Cloning • Microarrays • Chromatography • Immunochemistry • ELISA • Bioinformatics and techniques • Ethical issues

3. Assignment Presentation Quiz

4. Transfection : is the process of deliberately introducing nucleic acids into cells. Transformation: non-viral DNA transfer in bacteria, non-animal eukaryotic cells and plant cells. Transduction:is often used to describe virus-mediated DNA transfer. What to transfer Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Target: Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material.

5. TRANSFECTION  Transient Transfection Rapid, scalable, high-yield protein production from transiently transfected suspension cultures. the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded. Stable Transfection Stable transfection introduces DNA into cells long-term and pass the introduced DNA to their progeny. • marker gene is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain toxin

6. Common Marker Genes 

7. CHEMICAL-BASED TRANSFECTION • calcium phosphate, HEPES((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.

8. highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell.

9. Liposomes: small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. • Liposomes can be created by sonicating phosphatidylcholine rich phospholipids in water. Low shear rates create multilamellar liposomes, which have many layers like an onion. Continued high-shear sonication tends to form smaller unilamellar liposomes. In this technique, the liposome contents are the same as the contents of the aqueous phase. Sonication is generally considered a "gross" method of preparation as it can damage the structure of the drug to be encapsulated.

10. NON-CHEMICAL TRANSFECTION • Electroporation transient increase in the permeability of cell membrane is achieved when the cells are exposed to short pulses of an intense electric field. • Sonoporation uses high-intensity ultrasound to induce pore formation in cell membranes. • Optical transfection is a method where a tiny (~1 µm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser. • Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, or electroporation) are used in order to fuse the protoplast carrying the gene of interest with the target recipient cell. A major disadvantage of this method is that bacterial components are non-specifically introduced into the target cell as well. • Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is inserted into a cell. This approach can also be implemented with arrays of nanofibers that are introduced into large numbers of cells and intact tissue.

11. PARTICLE BASED TRANSFECTION • GENE GUN: where the DNA is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus.

12. Magnetofection, Nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid particle complexes towards and into the target cells, where the cargo is released.

13. Impalefection is carried out by impaling cells by elongated nanostructures and arrays of such nanostructures such as carbon nanofibers or silicon nanowires which have been functionalized with plasmid DNA. • particle bombardment. The nucleic acid is delivered through membrane penetration at a high velocity, usually connected to micro projectiles.

14. TRANSDUCTION  Introduced of DNA into cells using viruses as a carrier

15. Viral vectors are tailored to their specific applications but generally share a few key properties. • Safety: • modified in such a way as to minimize the risk of handling them by deletion of a part of the viral genome critical forviral replication. • Low toxicity: The viral vector should have a minimal effect on thephysiologyof the cell it infects. • Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design. • Cell type specificity: Most viral vectors are engineered to infect as wide a range ofcell typesas possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to bepseudo typed. • Identification: Viral vectors are often given certain genes that help identify which cells took up the viral genes.

16. APPLICATIONS Basic research Gene therapy vaccines

17. hTERT LNGFR Immortalization of primary T cells N S GP2-293 VIRAL VECTOR xlox(NGFR)TERT GP2xTERT11 PRODUCER CELL LINE ENVELOPE CONSTRUCT PACKAGED TERT RECOMBINANT VECTOR Staining with anti-NGFR ab beads PRIMARY T CELLS

18. Retroviruses • number of FDA-approved clinical trials such as the SCID-X1 trial. • either be replication-competent or replication-defective.. • involves the requirement for cells to be actively dividing for transduction. • cells such as neurons are very resistant to infection and transduction by retroviruses. • There is concern that insertional mutagenesis due to integration into the host genome might lead to cancer or leukemia

19. LENTIVIRUSES Lentiviruses are a subclass of Retroviruses. able to integrate into the genome of non-dividing cells, The site of integration is unpredictable, • studies have shown that lentivirus vectors have a lower tendency to integrate in places that potentially cause cancer ,clinical trials that utilized lentiviral vectors to deliver gene therapy for the treatment of HIV experienced no increase in mutagenic or oncologic events. • One or more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce the single-stranded RNA viral genome and is marked by the presence of the ψ (psi) sequence. This sequence is used to package the genome into the virion.

20. ADENOVIRUSES As opposed to Lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. • with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences. To overcome this problem scientists are currently investigating adenovirusesto which humans do not have immunity.

21. ADENO-ASSOCIATED VIRUSES AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy