CHAPTER 11

• Recombinant DNA technology

  The use of recombinant DNA technology has brought about significant advances in experimental genetics, gene mapping, and the diagnosis and treatment of disease

  
• Biotechnology industry


  The use of recombinant DNA technology has formed the basis for the biotechnology industry and the commercial production of human gene products for therapeutic uses, and the transfer of genes across species in agricultural plants and animals

  
• RFLP (Restriction Fragment Length Polymorphism) Mapping

  1. Genomic DNA extraction

  2. RE (Restriction Enzyme) digestion

  3. Gel electrophoresis for size fractionation

  4. Southern blotting to transfer DNA to nylon membrane

  5. Probe labelling and hybridisation of the labelled probe

  6. Pattern of polymorphism detected on X-ray film

  
• RFLP


  Detectable variations in DNA fragment length that are inherited as codominant alleles and can be used to follow the inheritance of individual chromosomes from generation to generation

  
• RFLP analysis

  • Based on restriction enzyme (RE) digestion of DNA and the transfer of DNA fragments to a filter, onto which can then be hybridised a labelled DNA fragment
  • RE commonly used for RFLP analysis recognizes 4-6 base-pair sequences, capable of reducing complex DNA, such as plant DNA, to a population of fragments with discrete sizes

  
• RFLP analysis of Huntington's Disease (HD)

  • Huntington's disease is an insidious disorder caused by an autosomal dominant mutation on short arm of chromosome 4
  • Occurs in about one of 10,000 individuals of European descent
  • Individuals with HD undergo progressive degeneration of the central nervous system, usually beginning at age 30 to 50 years and terminating in death 10 to 15 years later

  
• RFLP and Huntington's Disease


  The locus for the RFLP is closely linked to the locus for the disease-causing gene

  
• DNA fingerprinting


  A technology that identifies particular individuals using properties of their DNA

  
• DNA fingerprinting


  • Like the human fingerprint, the DNA of each individual is a distinctive characteristic that provides a means of identification
  • In forensics, samples of blood and semen can be subjected to DNA fingerprinting to determine whether a particular individual has been at a crime scene
  • DNA fingerprinting can determine if two individuals are related genetically

  
• Traditional DNA fingerprinting protocol

  1. Chromosomal DNA is isolated from a sample and digested with a restriction enzyme

  2. The resulting DNA fragments are then separated by gel electrophoresis

  3. The fragments in the gel are blotted on to a nitrocellulose filter, the DNA is denatured, and the filter is exposed to the radiolabeled probe

  4. The probe is complementary to a selected VNTR (Variable Number of Tandem Repeats) sequence, it hybridizes to approximately 5 to 30 fragments of DNA that contain this sequence and thereby labels 5 to 30 bands

  
• Automated DNA fingerprinting


  Uses PCR to amplify short tandem repeats (STRs), which are found in multiple sites in the genome of humans and other species and are variable among different individuals<|>The amplified STR fragments are fluorescently labeled and then separated by gel electrophoresis according to their molecular masses<|>A laser excites the fluorescent molecule within an STR, and a detector records the amount of fluorescence emission for each STR, yielded a series of peaks, each peak having a characteristic molecular mass

  
• DNA fingerprinting for identification and relationship testing

  • In medicine, it is used to identify different species of bacteria and fungi and even can distinguish among closely related strains of the same species
  • In forensics, it has provided critical evidence that an individual was at a crime scene
  • In relationship testing, persons who are related genetically will have some bands or peaks in common, and the number they share depends on the closeness of their genetic relationship

  
• Gene therapy


  The introduction of cloned genes into living cells in an attempt to cure disease

  
• Diseases targeted for gene therapy

  • Inherited human diseases involving a single gene abnormality (e.g. cystic fibrosis, sickle-cell anemia, hemophilia)
  • Diseases such as cancer and cardiovascular disease

  
• Nonviral gene transfer methods

  1. Involves the use of liposomes, which are lipid vesicles

  2. The DNA containing the gene of interest is complexed with liposomes that carry a positive charge

  3. The DNA-liposome complexes are taken up by cells via endocytosis
• Nonviral gene transfer


  • Advantage - liposomes do not elicit an immune response
  • Disadvantage - efficiency of gene transfer very low
• Viral gene transfer methods

  1. Involve the use of viruses

  2. The genetic modification of certain viral genomes has led to the development of virus vectors with a capacity to infect cells or tissues

  3. Gene therapy vectors have been genetically engineered so they have lost their capacity for replication in target cells

  4. Commonly used viruses for gene therapy include retroviruses, adenoviruses, and parvoviruses

  
• Viral gene transfer

  • Advantage - their ability to efficiently transfer cloned genes to a variety of human cell types
  • Disadvantage - the potential to evoke an undesirable immune response when injected into a patient. The inflammatory responses induced can be very severe

  
• Liposomes

  Lipid vesicles
• DNA-liposome complexes


  1. DNA containing the gene of interest is complexed with liposomes that carry a positive charge

  2. The DNA-liposome complexes are taken up by cells via endocytosis
• Liposomes


  • Do not elicit an immune response
• Nonviral gene transfers

  • Efficiency of gene transfer very low

  
• Viral gene transfers

  Involve the use of viruses

  
• Viral gene transfers

  1. The genetic modification of certain viral genomes has led to the development of virus vectors with a capacity to infect cells or tissues

  2. Gene therapy vectors have been genetically engineered so they have lost their capacity for replication in target cells

  
• Commonly used viruses for gene therapy

  • Retroviruses
  • Adenoviruses
  • Parvoviruses
• Viral gene transfers

  • Ability to efficiently transfer cloned genes to a variety of human cell types
  • Potential to evoke an undesirable immune response when injected into a patient. The inflammatory responses induced can be very strong and fatal to patients

  
• Adenosine deaminase (ADA)


  An enzyme involved in purine metabolism
• If both copies of the ADA gene are defective, deoxyadenosine will accumulate within the cells of the individual

  
• At high concentration, deoxyadenosine is particularly toxic to lymphocytes in the immune system, namely T cells and B cells
• The destruction of T and B cells leads to severe combined immunodeficiency disease (SCID)

  
• Treatments for SCID

  • Receive a bone marrow transplant from a compatible donor
  • Treat SCID patients with purified ADA that is coupled to polyethylene glycol (PEG)
  • Treat ADA with gene therapy
• The first SCID gene therapy

  1. Lymphocytes (i.e., T cells) were removed and cultured in a laboratory

  2. The lymphocytes were then transfected with a non-pathogenic retrovirus that had been genetically engineered to contain the normal ADA gene

  3. The retroviral genetic material is inserted into the host cell's DNA along with ADA gene carried by the virus

  4. The modified cells were reintroduced back into the patient

  
• T cells carrying the cloned gene were still detectable 8 to 10 years after they had been transferred

  
• However, most of the circulating T cells were not found to contain the cloned gene

  
• SCID-X1


  Inherited as an X-linked trait<|>Characterized by a block in T cell growth and differentiation<|>This block is caused by mutations in the gene encoding the c cytokine receptor, which plays a key role in the recognition of signals that are needed to promote the growth, survival, and differentiation of T cells

  
• Gene therapy trail for SCID-X1

  1. Normal c cytokine receptor gene was introduced into SCID-X1 patients

  2. At a 10-month follow up, T cells expressing the normal c cytokine receptor were detected in two patients and the cell counts had risen to normal level

  
• Molecular Pharming


  A novel avenue of research involves the production of medically important proteins in the mammary glands of livestock
• Human proteins successfully produced in the milk of domestic livestock


  • See Table 19.4

  
• Advantages of producing proteins in mammals vs bacteria

  • Certain proteins are more likely to function properly when expressed in mammals due to posttranslational modifications (e.g., attachment of carbohydrate groups) that occur in eukaryotes but not in bacteria
  • Certain proteins may be degraded rapidly or folded improperly when expressed in bacteria
  • The yield of recombinant proteins in milk can be quite large. A transgenic cow can produce approximately 1 g/L of the transgenic protein in milk

  
• Tissue-specific expression of the interested gene

  1. To produce a human gene into an animal so that the encoded protein will be secreted into its milk, the strategy is to clone the gene next to a milk-specific promoter

  2. Certain genes are expressed specifically within the mammary gland so that their protein product will be secreted into the milk

  3. Milk-specific genes include genes that encode milk proteins such as -lactoglobulin, casein, and whey acidic protein