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Vectors

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Vectors

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Vectors

  1. 1. 06/27/15 1
  2. 2. 06/27/15 2
  3. 3. Vectors can be defined in three ways: In epidemiology: An organism or vehicle that transmits the causative agent or disease- causing organism from the reservoir to the host. In molecular biology: A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism. In biology: A biotic agent that disperses reproductive structures of another organism, as a bee transmitting pollen to the stigma of a flower. 06/27/15 3
  4. 4. 06/27/15 4 Origin of replication Origin of replication Promoter Cloning site Protein purification tags Reporter genes: Reporter genes: Targeting sequence Antibiotic resistance Epitope Genetic markers
  5. 5. 06/27/15 5 Origin of replication Promoter Cloning site for replication and maintenance of vector in host cell. •to drive transcription of vector's transgene •Also to drive transcription of other genes in vector such as the antibiotic resistance gene •allow for the insertion of foreign DNA into the vector through ligation
  6. 6. 06/27/15 6 Protein purification tags Some expression vectors include proteins or peptide sequences that allows for easier purification of expressed protein. Reporter genes: Targeting sequence that allow for identification of plasmid that contains inserted DNA sequence. that directs the expressed protein to a specific organelle in the cell or specific location
  7. 7. 06/27/15 7 Antibiotic resistance EpitopeEpitope allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection. allows for antibody identification of cells expressing the target protein. Genetic markers Genetic markers allow for confirmation that the vector has integrated with the host genomic DNA.
  8. 8. Vectors perform their functions in two ways mostly: Transcription: Expression: 06/27/15 8
  9. 9. 2)Expression: • there are two types of expression vector: Prokaryotes expression vector: Eukaryotes expression vector: Prokaryotes expression vector: • Promoter • Ribosome Binding Site (RBS) • Translation initiation site - 06/27/15 9
  10. 10. 06/27/15 10 2)Expression: Eukaryotes expression vectors: These require sequences that encode for: Polyadenylation tail: Minimal UTR length: Kozak sequence:
  11. 11. In molecular biology, a vector may refer to oBinary vector oCloning vector oShuttle vector oExpression vector o Plasmid vector oViral vector 06/27/15 11
  12. 12. Plasmid: Plasmid can be defined as: An extra chromosomal self-replicating structure found in bacterial cells that carries genes for a variety of functions not essential for cell growth. OR A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA is called plasmid. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells.   06/27/15 12
  13. 13. 06/27/15 13 What are the main parts of a Plasmid? DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins Allows for selection of plasmid-containing bacteria. Allows for selection of plasmid-containing bacteria. Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a promoter. Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a promoter.  Drives transcription of target gene.  Vital component for expression vectors:  determines which cell types the gene is expressed in and amount of recombinant protein obtained.  Drives transcription of target gene.  Vital component for expression vectors:  determines which cell types the gene is expressed in and amount of recombinant protein obtained. Antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types. Gene, promoter or other DNA fragment cloned into the MCS for further study. Gene, promoter or other DNA fragment cloned into the MCS for further study. A short single-stranded DNA sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids. A short single-stranded DNA sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids.
  14. 14. What is mode of action of a plasmid vector?  Plasmids are pieces of DNA that can be manipulated through the use of restriction enzymes. Since these plasmids are already in the bacteria, the restriction enzymes are able to act as "molecular scissors" and cut up the part of the plasmid that is desired to be changed.  Then, the gene of interest is inserted as a new part of the plasmid, allowing the bacteria to now demonstrate this gene. 06/27/15 14
  15. 15. 06/27/15 15 What is mode of action of a plasmid vector? In the case of plasmids utilized as transcription vectors, incubating bacteria with plasmids generates hundreds or thousands of copies of the vector within the bacteria in hours, and the vectors can be extracted from the bacteria, and the multiple cloning sites can be cut by restriction enzymes to excise the hundredfold or thousand fold amplified insert. These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible.
  16. 16. What are the types of plasmid vectors? Plasmid vectors may be: Conjugative/transmissible vectors: They mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids. Non-conjugative vectors: They do not mediate DNA through conjugation, e.g., many R and col plasmids. 06/27/15 16
  17. 17. 1)Ti plasmid: A Ti or tumor inducing plasmid is a circular plasmid is often, a part of the genetic equipment of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Use to transduce its genetic material to plants. •Classification of Ti plasmid: •Based on the type of opine produced by their genes. The different opines specified by pTi are octopine, nopaline, succinamopine and leucinopine. •The plasmid has 196 genes that code for 195 proteins. There are no one structural RNA. The plasmid is 206,479 nucleotides long; the GC content is 56% and 81% of the material is coding genes. There are no pseudo genes. • Important in the creation of transgenic plants.06/27/15 17 What are the types of plasmid vectors?
  18. 18. 06/27/15 18 1)Ti plasmid:
  19. 19. 06/27/15 19 What are the types of plasmid vectors? 2)Col plasmid: Any plasmid that carries genetic information for the production of a colicin; Involved in killing the other harmful strains of bacteria also called bacteriocinogenic plasmid. They produce proteins that are sensitive to bacteria and inhibit its essential processes producing a clear area called lacuna. 3)Virulence Plasmid: Virulence plasmid turns the bacterium into a pathogen. So they are responsible for carrying the genes which cause diseases.
  20. 20. 06/27/15 20 What are the types of plasmid vectors?4)F Plasmid:  The Fertility factor allows genes to be transferred from one bacterium to another by conjugation. F plasmid a conjugative plasmid found in F+ (male) bacterial cells that leads with high frequency to its transfer. A cell possessing the F plasmid (F+ , male) can form a conjugation bridge (F pilus) to a cell lacking the F plasmid (F− , female), through which genetic material may pass from one cell to another.
  21. 21. 06/27/15 21 5)R (Resistance) Plasmid: What are the types of plasmid vectors? • R plasmid is a conjugative factor in bacterial cells. Promotes resistance to agents such as antibiotics, metal ions, ultraviolet radiation, and bacteriophage. • Many R-factors can pass from one bacterium to another and through which antibiotic resistance spreads between bacterial species, genera and even families. For example: RP1, a plasmid that encodes resistance to ampicillin, tetracycline and kanamycin originated in a species of Pseudomonas, but also in bacteria such as Escherichia coli.
  22. 22. 6)Degradative Plasmid: These plasmid are types of plasmids present in certain bacteria’s such as Pseudomonas putida which impart the ability of degrade xenobiotic compounds. For example: salicylic acid, 2-4D etc. There are 3 such plasmids: • CAMplasmid- which degrades camphor. • XYL, - , xylene. • NAH, - , naphthalene. 06/27/15 22 What are the types of plasmid vectors?
  23. 23. 06/27/15 23 What are the types of plasmid vectors? 7)Yeast plasmids: Yeast naturally harbors plasmids. These are 2µm plasmid - small circular plasmid often used for genetic engineering of yeast and linear pGKL plasmids from kluyveromyces lactis that are responsible for killer phenotype. Yeast cloning vectors include:
  24. 24. What is importance of Plasmids? Easy to work with - Plasmids are a convenient size (generally 1,000-20,000 base pairs). With current cloning technology, it is easy to create and modify plasmids containing the genetic element that you are interested in. Self-replicating - Once you have constructed a plasmid, you can make an endless number of copies of the plasmid by growing the plasmid in bacteria. Stable - Plasmids are stable long-term either as purified DNA or within bacteria (as glycerol stocks). Functional in many species and can useful for a diverse set of applications - Plasmids can drive gene expression in a wide variety of organisms, including plants, worms, mice and even cultured human cells. used to understand gene function, they can also be used to investigate promoters, small RNAs, or other genetic elements. 06/27/15 24
  25. 25. A viral vector is a virus which has been modified in a laboratory environment for purpose of introducing genetic material into a cell. 06/27/15 25 What are viral vectors?What are viral vectors?  Viruses are highly evolved natural vectors for the transfer of foreign genetic information into cells. But to improve safety, they need to be replication defective.  So the viruses can be used as vehicles to carry 'good' genes into a human cell.  To form a viral vector, remove the genes in the virus that cause disease. Then replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics). This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact.
  26. 26. 06/27/15 26 What are viral vectors?What are viral vectors?  Development of the viral vector dates to 1960s, when a number of researchers recognized that since viruses operate by inserting genetic material into cells, surely researchers could harness this trait by modifying viruses to change the genetic material they are inserting.  The process of delivering genetic material with the use of a virus is known as transduction, with the first successful attempt occurring in 1968 with plant cells.
  27. 27. 06/27/15 27 What are Key properties for a viral vector? Safety Low toxicity Stability Cell type specificity Identification
  28. 28. 06/27/15 28 What are Key properties for a viral vector? Safety  Viral vector should be modified in such a way as to minimize the risk of handling them.  Usually involves the deletion of a part of the viral genome critical for viral replication Low toxicity viral vector should have a minimal effect on the physiology of the cell it infects. Stability  Some viruses are genetically unstable & can rapidly rearrange their genomes.  This is detrimental to predictability and reproducibility of work conducted using a viral vector and is avoided in their design.
  29. 29. 06/27/15 29 What are Key properties for a viral vector? Cell type specificity  Most viral vectors are engineered to infect as wide a range of cell types as possible.  viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudo typed. Identification Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called Markers
  30. 30. 06/27/15 30 What are main types of viral vectors? Retrovirus Adenovirus Lentivirus Adeno-Associated virus (AAV) Herpes virus
  31. 31. 06/27/15 31  In Gene therapy  In vaccines production: Main applications of viral vectors?
  32. 32. 06/27/15 32 Main applications of viral vectors?  In Gene therapy:  Gene therapy is a technique for correcting defective genes responsible for disease development.  There are following delivery system for gene therapy: • Physical methods • Non-viral vectors • Viral vectors  Virus is usually “crippled” to disable its ability to cause disease and viral methods have proved to be the most efficient to date
  33. 33. 06/27/15 33 Main applications of viral vectors?  In Gene therapy:  If the pathogenicity of a specific virus, such as adenovirus, can be eliminated while the efficiency of gene transfer and expression is retained, the gene may be well suited for gene therapy.
  34. 34. 06/27/15 34 Main applications of viral vectors?  In vaccines production:  Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as DNA vaccines.  T-lymphocytes recognize cells infected with intracellular parasites based on the foreign proteins produced within the cell. T cell immunity is crucial for protection against viral infections and such diseases as malaria.  A viral vaccine induces expression of pathogen proteins within host cells. Since viral vaccines contain only a small fraction of pathogen genes, they are much safer and sporadic infection by the pathogen is impossible.  Adenoviruses are being actively developed as vaccines.
  35. 35. 06/27/15 35 This vaccine approach involves:  inserting influenza virus genes into a different carrier virus, or vector, that is used as a vaccine. DNA or RNA-encoding influenza proteins, such as hem agglutinin, are engineered into a vector that infects humans but does not cause disease. With a microbial vector vaccine, the vector itself, including the influenza genetic material, is injected directly into a person. The harmless vector virus can then express the proteins necessary to prompt an immune response.  In vaccines production:
  36. 36. 06/27/15 36 Retroviral Vectors:  Retrovirus Structure:  Outer coat:  Inner core:
  37. 37. 06/27/15 37 Lentivirus distinguishing features Presence of: Accessory genes ( regulation of transcription, RNA transport) Transduction in both dividing and non dividing cells Helper plasmids
  38. 38. 06/27/15 38 Retroviral mediated gene transfer: Transfection of packaging cell line:  Transfection of packaging cell line  Virus Collection  Transduction in target cells:  The production of replication defective virus is accomplished when packaging cells are transfected with plasmids that express all of the viral proteins necessary to generate infectious particles,  as well as the nucleic acid sequence of interest that will be packaged within them for delivery.  retroviral vectors produce replication defective, or self-inactivating, particles.  This allows for delivery of the desired sequence, without continued viral replication in the target cells.
  39. 39. 06/27/15 39  The packaging cells produce infectious particles, whose genome only encodes sequences from the transfer plasmid, which can be used to transduce the target cells.  The highest concentration of virus is typically produced between 48-72 hours.  To collect the virus, remove the supernatant from the packaging cell line.  The purified virus containing the desired gene can be stored at -80 °C until needed. Retroviral mediated gene transfer: 2-Virus Collection
  40. 40. 06/27/15 40 Retroviral mediated gene transfer: 2-Virus Collection
  41. 41. 06/27/15 41 3-Transduction in target cells  Viral Envelope glycoprotein protein interacts with the cellular receptor and enters the actively dividing cells.  The viral dsDNA of retroviruses is not capable of passing through the nuclear pore complex and requires breakdown of the nuclear membrane during mitosis for integration of nucleic acid.  The viral genome enters the nucleus and desired gene sequence is integrated into the host genome through the activity of restriction enzymes and Integrase while the other portion of viral genome is self inactivated and replication defective.  Some transfer vectors also contain a marker such as a fluorescent reporter or drug selection marker for the selection of transduced cells. Retroviral mediated gene transfer:
  42. 42. 06/27/15 42 3-Transduction in target cells Retroviral mediated gene transfer:
  43. 43. Comparison between bacterial & viral mediated gene transfer: Viral mediated  DNA insert length is upto 30kbp  More transfection effeciency  Long term persistence and stable gene transfer Bacterial mediated  DNA insert length is upto 10 kbp  comparitively less transfection effeciency 06/27/15 43
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