Biotech 2011-08-recombinant-dna


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Biotech 2011-08-recombinant-dna

  1. 1. Cloning Overview <ul><li>Foreign DNA is prepared for insertion into a vector DNA </li></ul><ul><ul><li>Both foreign and vector DNA are cut with a restriction enzyme </li></ul></ul><ul><ul><li>The restriction enzyme leaves sticky ends </li></ul></ul><ul><ul><ul><li>Short regions of homology at the site of cleavage </li></ul></ul></ul><ul><ul><li>The foreign DNA and vector DNA are mixed together </li></ul></ul><ul><ul><ul><li>The sticky ends bind DNA together </li></ul></ul></ul><ul><ul><li>DNA ligase reseals the double helices </li></ul></ul><ul><li>DNA is taken up by host cells </li></ul><ul><li>As the host cells are grown, the recombinant DNA grows with them </li></ul>
  2. 2. Libraries <ul><li>Libraries are made when DNA is cloned indiscriminately </li></ul><ul><ul><li>All possible DNA from a foreign source is inserted into vectors one fragment at a time </li></ul></ul><ul><ul><li>Bacteria take up the vectors en masse one vector per bacterium </li></ul></ul><ul><ul><li>Propagation of the bacteria creates a culture that contains all of the DNA from a foreign source, fragmented into small pieces, with each piece in its own bacterial clone </li></ul></ul>
  3. 3. Three general types of libraries <ul><li>Conceptually represent DNA, RNA and protein libraries </li></ul><ul><ul><li>Genomic libraries </li></ul></ul><ul><ul><ul><li>This is used to clone genes </li></ul></ul></ul><ul><ul><li>cDNA libraries </li></ul></ul><ul><ul><ul><li>Made from mRNA, this contains sequence representing message </li></ul></ul></ul><ul><ul><li>Expression libraries </li></ul></ul><ul><ul><ul><li>A cDNA library that expresses the foreign proteins </li></ul></ul></ul>
  4. 4. Genomic Library <ul><li>A genomic library contains all of the chromosomal DNA of a cell </li></ul><ul><li>DNA is purified and fragmented into pieces ranging from a few thousand bases to hundreds of thousands </li></ul><ul><ul><li>The size of the fragments depends on the capacity of the vector to contain and propagate the DNA </li></ul></ul><ul><ul><li>The fragments are ligated into a vector and the vector propagated in a suitable host cell culture </li></ul></ul><ul><ul><li>Each piece of chromosomal DNA is then grown within a foreign host cell </li></ul></ul>
  5. 5. cDNA Libraries <ul><li>A cDNA library contains the sequences of (primarily) mRNA found in a cell </li></ul><ul><ul><li>These sequences are propagated following conversion of a single stranded RNA molecule into double stranded DNA through the action of reverse transcriptase </li></ul></ul><ul><ul><li>They lack the transcriptional control and intronic sequences found in genomic clones </li></ul></ul><ul><ul><li>They are useful </li></ul></ul><ul><ul><ul><li>In understanding structure and function of an mRNA </li></ul></ul></ul><ul><ul><ul><ul><li>For example, the nucleotide sequence of an mRNA defines the exons of a gene </li></ul></ul></ul></ul><ul><ul><ul><li>In expressing eukaryotic proteins in bacteria </li></ul></ul></ul>
  6. 6. Expression libraries <ul><li>These are cDNA libraries in a special form of vector that permits transcription of the incorporated cDNA </li></ul><ul><ul><li>Proteins or protein fragments then appear with bacterial host cells that are normally not present </li></ul></ul><ul><ul><li>These can be identified based on their antigenicity or activities </li></ul></ul><ul><ul><li>By this route a cDNA sequence can be isolated based on identification of its protein product </li></ul></ul><ul><ul><li>Proteins can also be made in quantity and purified more easily when made in bacteria </li></ul></ul>
  7. 7. Why clone? <ul><li>Analytical purposes </li></ul><ul><ul><li>The DNA sequence and the structure/function relationships of a gene can be determined in isolation from the surrounding DNA of the genome by purifying the gene through cloning </li></ul></ul><ul><ul><ul><li>A single gene is lost in the background of the genome </li></ul></ul></ul><ul><ul><ul><li>Cloning it isolates it. </li></ul></ul></ul><ul><li>Practical purposes </li></ul><ul><ul><li>An isolated gene can be </li></ul></ul><ul><ul><ul><li>Expressed to produce a protein in vivo or in vitro </li></ul></ul></ul><ul><ul><ul><ul><li>Commercial purposes </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Therapeutic purposes </li></ul></ul></ul></ul><ul><ul><ul><li>Manipulated to change its sequence </li></ul></ul></ul><ul><ul><ul><ul><li>This makes new genes with original proteins and properties </li></ul></ul></ul></ul><ul><ul><li>Cloning creates an unlimited supply of identical copies </li></ul></ul>
  8. 8. Cloning reagents <ul><li>Enzymes and buffers </li></ul><ul><ul><li>Restriction enzymes </li></ul></ul><ul><ul><li>DNA ligase </li></ul></ul><ul><ul><li>And sometimes (for cDNA clones) </li></ul></ul><ul><ul><ul><li>Reverse transcriptase and its allied reagents </li></ul></ul></ul><ul><li>Vectors </li></ul><ul><li>Host cells </li></ul><ul><li>And </li></ul><ul><ul><li>Microbiological supplies </li></ul></ul><ul><ul><li>Radioactive or fluorescence labeled nucleotides </li></ul></ul>
  9. 9. DNA ligase <ul><li>While we have looked at ligase as an enzyme used during DNA replication, DNA ligase used during cloning procedures comes from the bacterial virus T4 </li></ul><ul><li>The enzyme is fragile and reactions are carried out at low temperature </li></ul><ul><li>It is capable of resealing the cut ends of restricted DNA, including blunt ends </li></ul><ul><li>Typically DNA is not purified away from restriction enzymes, but simply heated following restriction to denature the restriction enzyme </li></ul><ul><ul><li>Then ligation is not defeated by recutting </li></ul></ul>
  10. 10. Reverse transcriptase <ul><li>This is used for converting an RNA transcript into DNA such that it can be cloned. </li></ul><ul><ul><li>This is called copy DNA or cDNA </li></ul></ul><ul><li>mRNA is primed with oligo dT for reverse transcription </li></ul><ul><ul><li>RT makes a single stranded DNA copy of the RNA </li></ul></ul><ul><li>The RNA is completely degraded and several strategies are employed to prime second strand synthesis </li></ul><ul><ul><li>A hairpin loop naturally forms </li></ul></ul><ul><ul><ul><li>But this loses sequence on the 5’ end </li></ul></ul></ul><ul><ul><li>Oligo dA or dG can be enzymatically polymerized from the 3’ end </li></ul></ul><ul><ul><ul><li>Then oligo dT or dC can be used as with the first strand </li></ul></ul></ul>
  11. 11. <ul><li>The hairpin loop and ragged ends of the new duplex cDNA are digested with a single strand specific nuclease making the duplex blunt ended </li></ul><ul><li>Then “linker” DNA is ligated onto the duplex </li></ul><ul><ul><li>Linker DNA is a palindromic duplex oligonucleotide with a blunt end recognized by a restriction enzyme </li></ul></ul><ul><ul><ul><li>Here it is HindIII </li></ul></ul></ul><ul><ul><li>The linker DNA is then digested (with Hind III in this example) and the resulting DNA represents </li></ul></ul><ul><ul><ul><li>The mRNA </li></ul></ul></ul><ul><ul><ul><li>A Hind III sticky end </li></ul></ul></ul><ul><ul><li>This can be ligated into a vector as though it were genomic DNA that had simply been fragmented by Hind III </li></ul></ul>
  12. 12. Vectors <ul><li>DNA with an independent origin of replication and some selectable or differential markers </li></ul><ul><ul><li>A selectable marker permits a host cell to survive in otherwise lethal environments </li></ul></ul><ul><ul><li>A differential, non-selectable marker permits identification of a bacterium by its appearance </li></ul></ul><ul><ul><li>Plasmids, viruses (and viral derivatives) and artificial chromosomes </li></ul></ul><ul><ul><ul><li>All forms of cloning are technical variations on plasmid cloning </li></ul></ul></ul>
  13. 13. PBR322 <ul><li>This is an artificial plasmid vector that has educational value, but is rarely used anymore </li></ul><ul><ul><li>Commercial plasmid vectors are more versatile </li></ul></ul><ul><li>PBR322 has two genes for antibiotic resistance </li></ul><ul><ul><li>Amp and tet </li></ul></ul><ul><li>There is a single site for the restriction enzyme Bam H1 in the tet gene </li></ul><ul><ul><li>Inserting foreign DNA into this site inactivates the gene </li></ul></ul>
  14. 14. The host <ul><li>May be prokaryotic or eukaryotic </li></ul><ul><li>Must take up recombined DNA </li></ul><ul><ul><li>Technical approaches to introducing DNA into bacterial cells </li></ul></ul><ul><ul><ul><li>Add DNA to cells directly </li></ul></ul></ul><ul><ul><ul><ul><li>Transformation </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Bacteria take up plasmids </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><li>Transfection </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Bacteria take up viral vectors </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Eukaryotes take up any vectors </li></ul></ul></ul></ul></ul><ul><ul><ul><li>Electroporation </li></ul></ul></ul><ul><ul><ul><ul><li>Drive DNA into cells with electric field </li></ul></ul></ul></ul><ul><li>Must support growth of the recombined DNA </li></ul>
  15. 15. Colony screens <ul><li>Once recombinant DNA has been taken up by a host, a successful transformation needs to be identified </li></ul><ul><li>Bacterial colonies are grown on a nutrient plate </li></ul><ul><ul><li>If the foreign DNA was purified before it was inserted into the vector, then the selectable markers provide enough information to identify successful clones </li></ul></ul><ul><ul><li>However if the foreign DNA was not purified, then the bacterial colonies may overlayed with a membrane and lysed in situ on the membrane </li></ul></ul><ul><ul><ul><li>The bacteria and DNA will stick to the membrane </li></ul></ul></ul><ul><ul><ul><li>Successful clones hybridize to radioactive complementary DNA like a southern blot </li></ul></ul></ul>
  16. 16. What to do with the DNA? <ul><li>Once recombinant DNA is in a host, the host can be grown and plasmid easily isolated </li></ul><ul><li>Sequence it </li></ul><ul><ul><li>This can be done directly on purified recombinant DNA </li></ul></ul><ul><li>Express it </li></ul><ul><ul><li>Proteins may be made in quantity </li></ul></ul><ul><li>Mutate it </li></ul><ul><ul><li>Site specific mutations are possible </li></ul></ul><ul><ul><ul><li>Once a sequence is known, it is possible to alter any nucleotide by design </li></ul></ul></ul><ul><ul><ul><li>New proteins may be designed </li></ul></ul></ul><ul><ul><ul><li>Control elements of the inserted DNA may be studied and altered </li></ul></ul></ul>
  17. 17. Examples of medical relevance <ul><li>Insulin Dependent Diabetes Mellitus (diabetes type I) </li></ul><ul><ul><li>This results from an autoimmune destruction of the pancreatic beta cells </li></ul></ul><ul><ul><ul><li>The body can no longer make insulin </li></ul></ul></ul><ul><ul><li>Therapy requires monitoring of blood sugar and administration of insulin depending on glucose levels </li></ul></ul><ul><ul><ul><li>Insulin originally came from animal sources </li></ul></ul></ul><ul><ul><ul><ul><li>This molecule eventually elicited an immune response </li></ul></ul></ul></ul><ul><ul><ul><li>Cloning technology made it theoretically possible to clone human insulin </li></ul></ul></ul><ul><ul><ul><ul><li>There would be no immune response to this </li></ul></ul></ul></ul>
  18. 18. Cloning insulin <ul><li>The amino acid sequence of insulin was known, so a synthetic DNA sequence (probe) complementary to the insulin gene was available </li></ul><ul><ul><li>The gene sequence is not exactly predictable due to the degeneracy of the code, but close enough to insure a unique identification </li></ul></ul><ul><ul><li>The conditions at which probe is washed off of a membrane are made less harsh (lowering the stringency of the wash) </li></ul></ul><ul><ul><ul><li>This allowed imperfect hybrids of a sufficiently long probe to survive the wash </li></ul></ul></ul>
  19. 19. cDNA clones were necessary <ul><li>The insulin gene has two introns, one of which interrupts the coding sequence of the gene </li></ul><ul><li>In order to express the protein, a cDNA clone was needed that eliminated the intron </li></ul><ul><ul><li>Bacteria could not process mRNA from a cloned eukaryotic gene </li></ul></ul><ul><li>mRNA from an insulinoma (pancreatic beta cell tumor) was isolated and cDNA made </li></ul><ul><ul><li>This represented every message in the cell </li></ul></ul><ul><ul><ul><li>Insulin mRNA represented a fraction of the total </li></ul></ul></ul><ul><ul><li>All of the cDNA was inserted into plasmids at once </li></ul></ul><ul><ul><ul><li>It wasn’t possible to purify insulin mRNA first </li></ul></ul></ul><ul><ul><ul><li>Each vector got a cDNA from a random mRNA from the insulinoma </li></ul></ul></ul>
  20. 20. Identifying the insulin cDNA clone <ul><li>The culture of bacteria transformed with the insulinoma cDNA’s is called a library </li></ul><ul><ul><li>It represents all of the mRNA in the cell </li></ul></ul><ul><li>Bacteria were plated and the colonies “lifted” onto a membrane </li></ul><ul><ul><li>Probing the membrane with a synthetic complementary sequence identified colonies that contained the insulin cDNA </li></ul></ul>
  21. 21. Expressing the clone <ul><li>Once the cDNA was isolated, the “insert” was removed from the vector and cloned into a vector that contained control sequences for RNA polymerase </li></ul><ul><ul><li>The control sequences were the lac promoter </li></ul></ul><ul><ul><li>Transcription of the insulin gene could be increased with IPTG </li></ul></ul><ul><li>Bacteria were transformed with this recombinant vector and insulin protein was synthesized from the transcript polymerized by RNA polymerase </li></ul>
  22. 22. Problems with expressing eukaryotic genes in a bacterium <ul><li>But the insulin could not be properly processed in bacteria </li></ul><ul><ul><li>The signal peptide and the center peptide could not be removed by the bacterium </li></ul></ul><ul><ul><li>It was also difficult to purify because the signal peptide caused aggregation of the protein within the bacterium </li></ul></ul>
  23. 23. Another approach <ul><li>The A and B chains represented individual polypeptides that are normally produced by processing of preproinsulin </li></ul><ul><ul><li>It was necessary to remove the signal peptide from the clone prior to expression </li></ul></ul><ul><li>The gene was cloned as a fusion protein with beta galactosidase </li></ul><ul><ul><li>The gene, lacking the signal sequence but containing an N terminal methionine was used to replace the 3’ end of the betagalactosidase gene </li></ul></ul><ul><ul><li>This meant that expression of the cloned fusion gene produced a protein that was betagalactosidase on its amino terminal end and proinsulin (with an extra methionine) on the carboxyterminal end </li></ul></ul><ul><ul><ul><li>This helped in purification because beta galactosidase did not aggregate and was easily purified </li></ul></ul></ul><ul><ul><ul><li>But the insulin part had to be separated from beta galactosidase </li></ul></ul></ul>
  24. 24. Reconstitution of insulin from individual chains <ul><li>The extra methionine is specifically recognized and cleaved by the chemical reagent cyanogen bromide </li></ul><ul><ul><li>This splits the insulin away from the beta galactosidase </li></ul></ul><ul><li>Proinsulin was then mixed with C peptidases, that are made in pancreatic beta cells </li></ul><ul><ul><li>This freed the A and B chains (now linked through disulfide bridges) producing insulin </li></ul></ul>
  25. 25. Present day <ul><li>The complete cDNA (including signal peptide) have been cloned into yeast </li></ul><ul><li>The yeast contain ER and signal peptidase, and they have also been engineered to contain the protease that cleaves the internal c-peptide form insulin </li></ul><ul><ul><li>The yeast secrete human insulin </li></ul></ul><ul><ul><li>This circumvents the costly procedures necessary to purify insulin away from bacteria and then from the beta-gal. </li></ul></ul>
  26. 26. Gene therapy for IDDM type I? <ul><li>In immunodeficient mice, IDDM I can be cured with pancreatic beta cell transplantation </li></ul><ul><ul><li>But beta cells are killed by an autoimmune response in people </li></ul></ul><ul><ul><li>Transplanted beta cells would simply be killed by the immune system </li></ul></ul><ul><li>The use of insulin as a drug could be circumvented by putting an active insulin gene inside a patient </li></ul><ul><li>What kinds of problems might you expect in attempting this? </li></ul><ul><ul><li>How would you control the gene? </li></ul></ul><ul><ul><li>In what cell type would you put it? </li></ul></ul><ul><ul><li>What other systems in the host cell would be required for proper expression of insulin? </li></ul></ul>
  27. 27. Beta cell regulation <ul><li>The pancreatic beta cell responds to elevated blood glucose by releasing stored insulin through exocytosis </li></ul><ul><li>Properly regulated insulin requires </li></ul><ul><ul><li>Targeting to exocytic vesicles </li></ul></ul><ul><ul><ul><li>This is due to recognition of preproinsulin structure within the ER and Golgi </li></ul></ul></ul><ul><ul><ul><ul><li>Recognition systems must be present </li></ul></ul></ul></ul><ul><ul><ul><ul><li>The signal peptide and C-peptide must be removed </li></ul></ul></ul></ul><ul><ul><li>Storage prior to release </li></ul></ul><ul><ul><ul><li>Exocytic vesicles must form containing mature insulin </li></ul></ul></ul><ul><ul><ul><ul><li>They must be sequestered until a signal for release is received </li></ul></ul></ul></ul><ul><ul><li>Responsiveness of exocytosis to blood glucose </li></ul></ul><ul><ul><ul><li>Exocytosis involves elevated calcium levels that promote fusion of the exocytic vesicle with the plasma membrane </li></ul></ul></ul>
  28. 28. Other human diseases potentially amenable to gene therapy <ul><li>Most active </li></ul><ul><ul><li>Severe combined immune deficiency (SCID) </li></ul></ul><ul><ul><ul><li>Especially deficiency of adenosine deaminase </li></ul></ul></ul><ul><ul><ul><li>The expression of the gene needn’t be controlled and is expressed in rapidly growing cells (stem cells of the hematopoietic system) </li></ul></ul></ul><ul><ul><ul><li>Can’t find or transfect stem cells? </li></ul></ul></ul><ul><ul><li>Cystic fibrosis </li></ul></ul><ul><ul><ul><li>Lack of a chloride channel </li></ul></ul></ul><ul><ul><ul><li>Also expression needn’t be controlled </li></ul></ul></ul><ul><ul><ul><li>Accessible target cells (alveolar cells create the main clinical problem) </li></ul></ul></ul><ul><ul><ul><li>Delivery systems inadequate or unstable DNA? </li></ul></ul></ul><ul><li>Probability for a therapy to work increases if the expression levels of the protein don’t matter and that the defect is due to a missing enzyme </li></ul>