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Human Genetics - Chapter 19

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Human Genetics - Chapter 19 Powered By Docstoc
					Genetic & Reproductive
    Technologies

Objectives:
Define Biotechnology
Uses of Genetics in biotechnology
Applications of genetic technology
Reproductive technologies
                                     1
             Introduction
 Biotechnology is the use or alteration of
  cells or biological molecules for specific
  applications
 A transgenic organism has DNA from
  different species
 Recombinant DNA comes from more
  than one type of organism
 Both are possible because of the
  universality of the genetic code
                                               2
        Introduction




Mice containing the jellyfish gene for
  green fluorescent protein (GFP)
                                         3
          Amplifying DNA
 Polymerase chain reaction (PCR)
  – Works on DNA molecules outside of cells
  – Replicates sequence millions of times


 Recombinant DNA technology
  – Amplifies DNA within cells often using
    sequences from other organisms



                                              4
                   PCR
   Consists of a repetition of three basic
    steps:
     1. Denaturation: Heat is used to separate
     the two strands of target DNA
     2. Annealing: Two short DNA primers bind
     to the DNA at a lower temperature
     3. Extension: The enzyme Taq1 DNA
     polymerase adds bases to the primers
   All this is done in a thermal cycler
   Copies of DNA accumulate
    exponentially                                5
PCR




      6
7
Recombinant DNA Technology
    Recombinant DNA technology is also
     known as gene cloning
    It began in 1975 when molecular
     biologists convened to discuss the
     safety and implications of this new
     technology
    However, it turned out to be safer than
     expected
    – It also spread to industry faster and in more
      diverse ways than imagined
                                                      8
    Creating Recombinant DNA
            Molecules
   Manufacturing recombinant DNA
    requires restriction enzymes that cut
    donor and recipient DNA at the same
    sequence
   These enzymes cut DNA at sites that
    are palindromic
   The cutting action of many of these
    enzymes generate single-stranded
    extensions called “sticky ends”         9
10
Creating Recombinant DNA
        Molecules
   Another “tool” used is a cloning
    vector
    – Carries DNA from the cells of one species
      into the cells of another

   Commonly used vectors include:
    – Plasmids
    – Bacteriophages
    – Disabled retroviruses
                                                  11
Creating Recombinant DNA
        Molecules
   Cut DNA from donor and plasmid
    vector with the same restriction
    enzyme
   Mix to generate recombinant DNA
    molecule
   When such a modified plasmid is
    introduced into a bacterium, it is mass
    produced as the bacterium divides
                                              12
13
     Isolating Gene of Interest
   Genomic library: Collections of
    recombinant DNA that contain pieces of
    the genome
   DNA probe: Radioactively (or
    fluorescently) labeled gene fragments
    – “Green mice”
   cDNA library: Genomic library of
    protein encoding genes produced by
    extracting mRNA and using reverse
    transcriptase to make DNA
                                             14
15
Selecting Recombinant Molecules
    Three types of recipient cells can result
     from attempt to introduce a DNA
     molecule into a bacterial cell
     1. Cells that lack plasmids
     2. Cells with plasmids that do not contain
      foreign genes
     3. Cells that contain plasmids with foreign
      genes


                                                   16
Selecting For Cells With Vectors
    Vectors are commonly engineered to
     carry antibiotic resistance genes
    Host bacteria without a plasmid die in
     the presence of the antibiotic
    Bacteria harboring the vector survive
    Growing cells on media with antibiotics
     ensures that all growing cells must carry
     the vector
                                             17
Selecting For Cells With Inserted DNA

     The site of insertion of the DNA of
      interest can be within a color-
      producing gene on the vector

     Insertion of a DNA fragment will
      disrupt the vector gene
      – And so the bacterial colony that grows will
        be colorless

                                                      18
Applications of Recombinant DNA
     Recombinant DNA is used to:
     – Study the biochemical properties or genetic
       pathways of that protein
     – Mass-produce proteins (e.g., insulin)
     Sometimes conventional methods are still the
      better choice because of economics
     Textile industry can produce indigo dye in E.
      coli by genetically modifying genes of the
      glucose pathway and introducing genes from
      another bacterial species
                                                      19
20
          Transgenic Animals
    An even more efficient way to express
     some recombinant genes is in a body
     fluid of a transgenic animal

    Transgenic sheep, cows, and goats
     have all expressed human genes in their
     milk,
    – Clotting factors
    – Clot busters
    – Collagen
    – Antibodies                               21
    Several techniques are used to insert
     DNA into cells to create transgenic
     animals
    – Chemicals that open transient holes in
      plasma membrane
    – Liposomes that carry DNA into cells
    – Electroporation: A brief jolt of electricity to
      open membrane
    – Microinjection: Uses microscopic needles
    – Particle bombardment: a gun like device
      shoots metal particles coated with foreign
      DNA

                                                        22
         Transgenic Animals
   Finally, an organism must be
    regenerated from the altered cell

   If the trait is dominant, the transgenic
    animal must express it in the appropriate
    tissue at the right time in development

   If the trait is recessive, crosses between
    heterozygotes may be necessary to yield
    homozygotes that express the trait
                                                 23
              Animal Models
   Transgenic animals are far more useful
    as models of human diseases
    – Example: Inserting the mutant human beta
      globin gene that causes sickle-cell anemia
      into mice

   Drug candidates can be tested on these
    animal models before testing on humans
    – Will be abandoned if they cause significant
      side effects
                                                    24
              Animal Models
   Transgenic animal models have
    limitations
    – Researchers cannot control where a
      transgene inserts, and how many copies do
      so
    – The level of gene expression necessary for a
      phenotype may differ in the model and
      humans
    – Animal models may not mimic the human
      condition exactly because of differences in
      development or symptoms
                                                     25
Animal Models




                26
              Bioremediation
   Transgenic organisms can provide
    processes as well as products

   Bioremediation: The use of bacteria or
    plants to detoxify environmental pollutants

   Examples
    – Nickel-contaminated soils
    – Mercury-tainted soils
    – Trinitrotoluene (TNT) in land mines
                                              27
    Monitoring Gene Function
   Gene expression DNA microarrays (gene
    chips) are devices that detect and display
    the mRNAs in a cell
   A microarray is a piece of glass or plastic
    that is about 1.5 centimeters square
   Many small pieces of DNA of known
    sequence are attached to one surface, in a
    grid pattern
   In many applications, a sample from an
    abnormal situation is compared to a normal
    control
                                                  28
    Monitoring Gene Function
   Messenger RNAs are extracted from
    the samples and cDNAs are made
   These are differentially-labeled and
    then applied to the microarray
   The pattern and color intensities of the
    spots indicate which genes are
    expressed
   A laser scanner detects and computer
    algorithms interpret the results
                                               29
Monitoring Gene Function




                           30
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32
33
             Silencing DNA
   In some situations, silencing gene
    expression may be useful
    – Blocking transcription of oncogenes


   Three techniques can be used to control
    gene expression
    – RNA interference
    – Antisense sequences
    – Knockouts from gene targeting
                                            34
Knockouts from Gene Targeting
   Gene targeting is a technique that uses
    homologous recombination to replace a
    normal DNA sequence with one that
    cannot be transcribed or translated
    – This silences gene expression by creating a
      “knockout” gene
    – Moreover, observing what happens (or not)
      can reveal the gene’s normal function
   A variation of the technique exchanges
    genes that have an altered function,
    producing a “knockin”
                                                    35
Knockouts from Gene Targeting

   Knockout mice are valuable in several
    ways:

    – Are more accurate models than transgenic
      mice
    – Populations are easily tested
    – Knockouts for several genes can be created
      to observe polygenic traits
    – Mice with diseases that humans also get can
      be observed
                                                    36
    Treating Genetic Disease
 Treatments have evolved through stages
     1) Removing an affected body part
     2) Replacing an affected body part or
  biochemical with material from a donor
     3) Delivering pure, human proteins
  derived from recombinant DNA technology
  to compensate for the effects of a mutation
     4) Gene therapy, to replace mutant
  alleles
                                                37
          Gene Therapy
 Altering genes theoretically can provide
  a longer-lasting effect than treating
  symptoms
 The first efforts focused on inherited
  disorders with a known mechanism,
  even though the conditions are rare
 Gene therapy now is targeting more
  common illnesses, such as heart
  disease and cancers
                                             38
          Gene Therapy
 Germline gene therapy
    - Gamete or zygote alteration;
  heritable; not done in humans; creates
  transgenic organisms

 Somatic gene therapy
    - Corrects only the cells that a
  disease affects; not heritable
                                           39
40
41
       Assisted Reproductive
           Technologies
 ARTs are methods that replace the source of a
  male or female gamete, aid fertilization or
  provide a uterus
 Developed to treat infertility but are becoming
  part of genetic screening
 The US Government does not regulate ARTs
     - However, the British Government does


                                                    42
   Infertility and Subfertility
 Infertility is the inability to conceive a
  child after a year of frequent intercourse
  without contraceptives
 Subfertility distinguishes couples who
  can conceive, but require longer time than
  usual
 Affect one in six couples
 A physical cause can be identified in 90%
  of cases: 30% in males, 60% in females
                                               43
          Male Infertility
 One in 25 men are infertile
 Easier to detect, but often harder to treat
  than female infertility
 Most cases of male infertility are genetic
 Causes of infertility include:
  – Low sperm count (oligospermia)
  – A malfunctioning immune system
  – A varicose vein in the scrotum
  – Structural sperm defects
                                            44
Male Infertility




                   45
 Most cases of male infertility are genetic
  – Due to small deletions of Y chromosome that
    remove genes important for spermatogenesis
  – Mutations in genes for androgen receptors or
    other hormones promoting sperm
    development
 In cases of low sperm count, sperm can
  be stored frozen, then pooled
 Lack of motility in sperm prevents
  movement in the female reproductive tract

                                                   46
          Female Infertility
 Many women with subfertility or infertility
  have irregular menstrual cycles
  – This makes it difficult to pinpoint when
    conception is most likely
 Tracking ovulation cycles aids in
  determination of the most likely days for
  conception
 Abnormalities in any part of the female
  reproductive system can cause infertility
                                                47
48
         Female Infertility
 Fertility drugs stimulate ovulation but
  may induce release of multiple oocytes
 Blocked fallopian tubes can result in
  ectopic pregnancy (tubal pregnancy).
 Excess tissue growth in uterine lining
  may make it inhospitable for an embryo
  – Fibroids: benign tumors
  – Endometriosis: buildup of uterine lining

                                               49
         Female Infertility
 Secretions in the vagina and cervix may
  be hostile to sperm
 Infertility may also result if the oocyte
  fails to release sperm-attracting
  chemicals
 Early pregnancy loss due to an abnormal
  chromosome number is more common in
  older females
  – May appear as infertility because bleeding
    resembles a heavy menstrual flow
                                                 50
           Infertility Tests
 The man is checked first, because it is
  easier, less costly and less painful to
  obtain sperm than oocytes
  – Sperm are checked for number (sperm
    count) motility and morphology (shape)
 A gynecologist can then check the
  female to see if reproductive organs are
  present and functioning
 Psychological factors may also be at
  play
                                             51
     Assisted Reproductive
     Technologies (ARTs)
 Many people with fertility problems use
  alternative ways to conceive

 In the US, about 1% of the 4 million
  births each year are from ARTs

 Several of the ARTs were developed in
  nonhuman animals
                                            52
     Assisted Reproductive
     Technologies (ARTs)
 Examples
    - Intrauterine insemination
    - Surrogate motherhood
    - In vitro fertilization (IVF)
    - Gamete intrafallopian transfer (GIFT)
    - Zygote intrafallopian transfer (ZIFT)
    - Oocyte banking and donation
    - Preimplantation genetic diagnosis
                                               53
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     Intrauterine Insemination
 Donated sperm is placed in a woman’s
  reproductive tract, typically at the cervix or in
  uterus
 Success rate is 5-15%
 1790: first reported pregnancy from artificial
  insemination
 1953: methods for freezing and storing sperm
  were developed
 Sperm catalogs list personal characteristics

                                                      56
     Surrogate Motherhood
 In surrogate motherhood, a woman
  carries a pregnancy to term for another
  woman who cannot conceive and/or
  carry the pregnancy
 Custody rights are given up at birth
 A surrogate mother may or may not have
  contributed an oocyte
 Complex legal and emotional issues
  must be considered

                                            57
     In vitro Fertilization (IVF)
 For in vitro fertilization, a sperm fertilizes an
  oocyte in a culture dish
 Embryos are transferred to the oocyte donor’s
  uterus (or a surrogate’s uterus) for implantation
 1978: First IVF child born (Louise Joy Brown)
     - Since then, 4 million IVF children
 Intracytoplasmic sperm injection (ICSI) is more
  effective than IVF alone


                                                      58
Intracytoplasmic Sperm Injection
 For cases in which sperm cannot
  penetrate the oocyte, IVF can be
  accompanied by ICSI which injects
  sperm directly into the oocyte
 ICSI allows conception in cases of low
  sperm count, abnormal sperm shape,
  sperm motility problems


                                           59
Intracytoplasmic Sperm Injection




                                   60
Gamete Intrafallopian Transfer
           (GIFT)
 GIFT is a method in which superovulated
  oocytes from a woman and sperm from
  her partner are placed together in her
  uterine (fallopian) tube
 Fertilization occurs in the woman’s body
 Allows conception in cases of fallopian
  tube blockage
 22% success rate and costs less than IVF
                                             61
Zygote Intrafallopian Transfer
            (ZIFT)
 IVF ovum is introduced into the uterine
  tube and allowed to move to the uterus
  for implantation
 Also about 22% successful
 GIFT and ZIFT are done much less
  frequently than IVF
     - They often will not work for women
  with scarred uterine tubes
                                             62
    Oocyte Banking and Donation
 Oocytes, like sperm, can be stored frozen
 Only 3% successful
 New technique can freeze strips of ovarian tissue
 Difficulties because oocytes pause in meiosis II
  until fertilization occurs
 Women can store their own oocytes to have
  children later or prior to undergoing chemotherapy
 Donated oocytes can be used by women with
  infertility problems; 28-50% successful
 Embryo adoption is a variation on oocyte donation
                                                   63
Preimplantation Genetic Diagnosis
              (PGD)
   This PGD technique allows detection of
    genetic and chromosomal abnormalities
    prior to implantation
   One cell or blastomere of an 8-celled
    embryo can be removed for testing
       - The remaining cells will complete
    normal development
   About 29% success rate
                                              64
Preimplantation Genetic Diagnosis
              (PGD)
   1989: First children who had PGD
      - Used to select females who could not
    inherit X-linked disease from mother
   1992: First child born following PGD to
    screen for cystic fibrosis allele present in
    her family
   PGD can be combined with IVF for
    women who have had multiple
    miscarriages
                                               65
Preimplantation Genetic Diagnosis
              (PGD)




                                66
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         Extra Embryos
 Sometimes ARTs leave “extra” oocytes,
  fertilized ova, or very early embryos




                                          68
Using Extra
 Embryos




              69
           Extra Embryos
 A similar case to the Lyons’ involved a
  California woman named Nadya Suleman
     - She had eight fertilized ova left over
  after using six to produce her six young
  children
     - She did not want to destroy these
  ova or continue to store them
     - She was implanted with them, and in
  early 2009 gave birth to octuplets!
                                                 70
        Polar Body Biopsy
 An experimental ART that increases the
  success of IVF
 Based on Mendel’s first law (segregation
  of alleles)
 Enables physicians to perform genetic
  tests on polar bodies and infer the
  genotype of the accompanying oocyte
 Oocytes that pass this test can be
  fertilized in vitro and the resulting embryo
  implanted
                                             71
Polar Body Biopsy




                    72
Assisted Reproductive Disasters
  ARTs introduce ownership and
   parentage issues

  Another controversy is that human
   genome information is providing more
   traits to track and perhaps control in
   coming generations
      - So, who will decide which traits are
   worth living with, and which are not?
                                                73
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