Human Genetics - Chapter 19 by niusheng11


									Genetic & Reproductive

Define Biotechnology
Uses of Genetics in biotechnology
Applications of genetic technology
Reproductive technologies
 Biotechnology is the use or alteration of
  cells or biological molecules for specific
 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

Mice containing the jellyfish gene for
  green fluorescent protein (GFP)
          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

   Consists of a repetition of three basic
     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

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
    However, it turned out to be safer than
    – It also spread to industry faster and in more
      diverse ways than imagined
    Creating Recombinant DNA
   Manufacturing recombinant DNA
    requires restriction enzymes that cut
    donor and recipient DNA at the same
   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
Creating Recombinant DNA
   Another “tool” used is a cloning
    – Carries DNA from the cells of one species
      into the cells of another

   Commonly used vectors include:
    – Plasmids
    – Bacteriophages
    – Disabled retroviruses
Creating Recombinant DNA
   Cut DNA from donor and plasmid
    vector with the same restriction
   Mix to generate recombinant DNA
   When such a modified plasmid is
    introduced into a bacterium, it is mass
    produced as the bacterium divides
     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
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

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
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

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
          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
    – Clotting factors
    – Clot busters
    – Collagen
    – Antibodies                               21
    Several techniques are used to insert
     DNA into cells to create transgenic
    – 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

         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
              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
              Animal Models
   Transgenic animal models have
    – Researchers cannot control where a
      transgene inserts, and how many copies do
    – The level of gene expression necessary for a
      phenotype may differ in the model and
    – Animal models may not mimic the human
      condition exactly because of differences in
      development or symptoms
Animal Models

   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
    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
    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
   A laser scanner detects and computer
    algorithms interpret the results
Monitoring Gene Function

             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
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”
Knockouts from Gene Targeting

   Knockout mice are valuable in several

    – Are more accurate models than transgenic
    – 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
    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
          Gene Therapy
 Altering genes theoretically can provide
  a longer-lasting effect than treating
 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
          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
       Assisted Reproductive
 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

   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
 Affect one in six couples
 A physical cause can be identified in 90%
  of cases: 30% in males, 60% in females
          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
Male Infertility

 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
 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

          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
 Abnormalities in any part of the female
  reproductive system can cause infertility
         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

         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
 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
           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
     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
     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
     Intrauterine Insemination
 Donated sperm is placed in a woman’s
  reproductive tract, typically at the cervix or in
 Success rate is 5-15%
 1790: first reported pregnancy from artificial
 1953: methods for freezing and storing sperm
  were developed
 Sperm catalogs list personal characteristics

     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

     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

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

Intracytoplasmic Sperm Injection

Gamete Intrafallopian Transfer
 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
Zygote Intrafallopian Transfer
 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
    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
Preimplantation Genetic Diagnosis
   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
Preimplantation Genetic Diagnosis
   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
Preimplantation Genetic Diagnosis

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

Using Extra

           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
     - 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!
        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
Polar Body Biopsy

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?

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