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Biotech 2011-08-recombinant-dna
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    Biotech 2011-08-recombinant-dna Biotech 2011-08-recombinant-dna Presentation Transcript

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