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Title: Animal biotechnology lecture 2


1
Animal biotechnology lecture 2
  • Dr. Ziad Jaradat

2
Animal Biotechnology Transgenic Animals
  • Since the early 1980s, fruit flies, fish, sea
    urchins, frogs, laboratory mice and farm animals,
    such as cows, pigs, and sheep have been
    successfully produced.
  • The ability to manipulate the genome of the whole
    animal and the production of transgenic animals
    has influenced the science dramatically in the
    last 15 years.
  • The procedure for introducing exogenous donor DNA
    into a recipient cell is called Transfection.
  • Chromosomes are taken up inefficiently so that
    intact chromosomes rarely survived the procedure.
    Instead the recipient cell usually get a part of
    the DNA.

3
  • Now, with the advent of the recombinant DNA, the
    possibility of introducing a particular segment
    of DNA become possible. However, still there are
    always some problems of the stability of the new
    inserts (transient transfectants).
  • An exciting development of transfection
    techniques is the application of DNA technology
    to introduce genes into animals.

4
  • An animal that gains new genetic information from
    the addition of foreign DNA is described as
    Transgenic while the introduced DNA is called the
    transgene.
  • The transgenes are introduced into the pronuclei
    of fertilized eggs by injection, and the injected
    embryos are incubated in vitro or implanted into
    the uterus of a pseudopregnant female for
    subsequent development.
  • What is Pronucleus? For a short time after
    fertilization, the male pronucleus and female
    pronucleus exist separately.
  • Female pronucleus In the maturing of the ovum
    preparatory to impregnation, a part of the
    germinal vesicle becomes converted into a number
    of small vesicles, which aggregate themselves
    into a single clear nucleus which travels towards
    the center of the egg and is called the female
    pronucleus.

5
  • Male pronucleus In impregnation, the spermatozon
    which enters the egg soon loses its tail, while
    the head forms a nucleus, called the male
    pronucleus, which gradually travels towards the
    female pronucleus and eventually fuses with it,
    forming the first segmentation nucleus. The male
    pronucleus is larger than the females and can be
    seen fairly easily under a light microscope.

6

7
  • Synopsis of the transgenesis process
  • Plasmids carrying the gene of interest are
    injected into the germinal vesicle (nucleus) of
    the oocyte or into the pronucleus (before uniting
    with the gamete) of the fertilized egg.
  • The egg is implanted into a pseudopregnant mouse
  • After birth, the recipient mouse can be examined
    to see whether it has gained the foreign DNA and
    if so whether it is expressed.
  • As a result multiple copies of transgenes are
    integrated at random locations in the genome of
    the transgenic individuals.
  • The transgenes in many transgenic individuals are
    also transmitted through the germline to
    subsequent generations.

8
  • Note If the transgenes are linked with
    functional promoters, expression of transgenes as
    well as display of change in phenotype is
    expected in some of the transgenic individuals
  • Questions to be asked about any transgenic animal
    are
  • how many copies it has of the foreign DNA (varies
    1-50)

9
  • where these copies are located usually multiple
    copies are integrated into a tandem array
    (arranged adjacent to each other) into a single
    chromosomal site
  • whether they are present in the germ line and
    inherited in Mendelian manner.
  • can the gene be expressed independently? i.e does
    the regulatory elements function independently
  • are transfected genes expressed with the proper
    developmental specificity?
  • A good result if we obtain 15 of the animals to
    be transgenic.
  • In the progeny of the infected animal, the
    expression of the donor gene is extremely
    variable and that could be dependent on the place
    of integration of the new DNA.

10
  • Transgenesis Methodology
  • Transgenic technology has been developed and
    perfected in the laboratory mouse. Since the
    early1980s hundreds of different genes have
    been introduced into various mouse strains. These
    studies have contributed to
  • understanding of gene regulation
  • tumor development, example introducing oncogenes
    and observe the effect

11
  • immunological specificity, example producing
    knockout genes that are responsible for some
    immunological aspects
  • molecular genetics of development
  • other biological interests such as examining the
    possibility of using transgenic animals in the
    industrial production of human therapeutic
    drugs.. etc.

12
  • Methods of gene transfer in animals
  • For transgenesis, DNA can be introduced into mice
    by one of the following methods
  • Retroviral vectors that infects the cells of an
    early stage embryo prior to implantation into a
    receptive female.
  • Microinjection into the enlarged sperm nucleus
    (the male pronucleus) of a fertilized egg
  • Introduction of genetically engineered embryonic
    stem cells into an early stage developing embryo
    prior to implantation into a receptive female.
  • Transfer of diploid somatic nuclei into an
    enucleated oocyte.

13
  • Retrovirus-Mediated Gene Transfer
  • The most useful vectors for the purpose of gene
    isolation are those that lend themselves to the
    production of libraries consisting of overlapping
    fragments of genomic DNA, ideally encompassing
    the entire genome several times.
  • Exmaple bacteriophage ? genomic library of 106
    viruses each containing on average 20 Kb of DNA,
    represents 6-7 copies of the entire mouse genome
    and the probability that each gene is represented
    is very high.
  • Retroviruses can be used for the transfer of
    foreign genes into animal genomes.

14
  • This can best be done at 4-16 cell stage
    embryos. However, it can be done up to
    midgestation, but with incomplete infections i.e
    low infectivity rate.
  • Immediately following infection, the retrovirus
    produces a DNA copy of its RNA genome using its
    reverse transcriptase.
  • Completion of this process requires that the host
    cell undergoes the S phase of the cell cycle.
    Therefore, retroviruses effectively transduce
    only mitotically active cells.
  • Modifications to the retrovirus frequently
    consist of removal of structural genes, such as
    gag, pol, and env, which support viral particle
    formation.

15
  • Additionally, most retroviruses and complementary
    lines are ecotropic in that they infect only
    rodents, such as rats and mice, and rodent cell
    lines rather than humans.
  • The DNA copy of the viral genome, or provirus,
    integrates randomly into the host cell genome,
    usually without deletions or rearrangements.
  • Because integration is not by way of homologous
    recombination, this method is not used
    effectively for site-directed mutagenesis.
  • Very high rates of gene transfer are achieved
    with the use of retroviruses.

16

Table of common vectors used for such purpose
  • Vector Origin Insert
    size range Multicopy plasmids multicopy plasmids
    up to 20 kb
  • Lambda vectors Bacteriophage ? up to 30 kb
  • Cosmid Bacteriophage ? up to 40 kb
  • P1 artificial chrom Bacteriophage P1
    80-90 kb
  • Bacterial artificial chrom. Large Bacteria
    plasmid 100-300 kb (F factor)
  • Yeast chrom. (YAC) Yeast chromosome
    100-1000 kb
  • means indefinite.

17
  • Disadvantages of this method include
  • Low copy number integration.
  • Additional steps required to produce
    retroviruses.
  • Limitations on the size of the foreign DNA insert
    (usually 9 to 15 kb) transferred.
  • Potential for undesired genetic recombination
    that may alter the retrovirus.
  • High frequency of mosaicism.
  • Possible interference by integrated retroviral
    sequences in transgene expression.

18
  • The genome of the retroviral strain can be
    integrated into the same nucleus as the
    transgene. This means that the virus itself could
    be produced by the transgenic organism and create
    a problem especially if the animal will be used
    for production of food.
  • Also the provirus attracts methylation which
    possibly in conjugation with other mechanisms
    disables its expression when it passes through
    the germ line.
  • Due to this, and to the availability of other
    alternative methods, the retroviral vector method
    is rarely used for producing transgenic animals
    that have a commercial potential.

19
DNA Microinjection Method
  • Because of the disadvantages of the retroviral
    vectors, microinjection of DNA is currently the
    preferred method for producing transgenic mice.
  • First - you need the gene of interest in the
    proper form. A linear transgene construct is
    made, which contains
  • the structural gene of interest, with introns
  • a strong mouse gene promoter and enhancer to
    allow the gene to be expressed
  • vector DNA to enable the transgene to be inserted
    into host DNA
  • The immature female mice will be induced to
    superovulate by sequential administration of
    FSH/LH and HCG and mated to fertile males.
    One-celled embryos are flushed from the oviducts
    and placed in a drop of medium and viewed by
    phase-contrast or interference microscopy.

20
  • This procedure has the following steps
  • The number of available fertilized eggs that are
    to be inoculated are increased by stimulating
    donor females to superovulate.
  • This can be done by
  • Giving the mice an initial injection of pregnant
    mares ( an adult female of horse or related
    mammal) serum
  • Another injection about 48 hours later of human
    chorionic gonadotropin (hCG). By this protocol
    the female produces about 35 eggs instead of the
    normal number of 5-10.

21
  • These females are mated, then sacrificed and the
    fertilized eggs (oocytes) are flushed from their
    oviducts and recovered.
  • Eggs are treated with hyaluronidase to remove
    adherent follicle cells.
  • Unfertilized eggs are discarded
  • The eggs are inoculated immediately with the
    transgene, briefly
  • embryo at the pronuclear stage is held in place
    by suction.
  • a micro needle loaded with a suspension of
    plasmid DNA will be prepared.
  • It is introduced through the zona pellucida and
    plasma membrane into the most accessible
    pronucleus (usually the male) and
  • several hundred molecules of the recombinant DNA
    are injected in a volume of approximately 1
    picoliter (p1).
  • on a good day several hundred eggs can be
    injected.
  • The male pronucleus can be located by using
    dissecting microscope and the eggs then can be
    maneuvered, oriented and held in place while the
    DNA is microinjected.

22
oocyte
Pippet
Micro needle
23
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24
  • After inoculation, 25-40 eggs are implanted
    microscopically into a foster mother who has been
    made pseudo-pregnant by being mated to a
    vasectomized male so that none of the eggs of the
    foster mother will be fertile therefore, the
    foster mother will deliver pups from the
    implanted fertile eggs three weeks after the
    inoculation.
  • After birth, the presence of foreign material is
    studied by DNA hybridization with appropriate
    probes or PCR.
  • A transgenic mouse can be mated to another to
    produce transgenic homozygous transgenic animal.

25
  • More details on gene transfer.

26
Gene Transfer to Animal cells
  • Dr. Ziad Jaradat

27
  • There are four major strategies for gene transfer
    to animal cells, two of which are considered
    biological mechanisms using virus (transduction)
    or bacterial that invade animal cells
    (bactofection), those methods involve infection
    while the other two are chemical and physical
    methods which do not involve infection thus
    termed transfection.
  • Virus (transduction) the transferred gene
    represents part of the viral genome
  • Bacteria (bactofection) the gene will be
    transferred as a plasmid

28
Chemical transfection
  • DNA will be taken from the surroundings when
    the DNA is presented as a synthetic complex
    either as
  • a complex with overall positive charge, allowing
    it to interact with negatively charge cell
    membrane and promote uptake by endocytosis
  • as lipophilic complex that fuses with the cell
    membrane and deposits the transgene directly into
    the cytoplasm.

29
Physical transfection
  • in this method, naked DNA is deposited directly
    into the cell by exploiting a physical force.
    This includes
  • microinjection
  • particle bombardment
  • ultrasound
  • electroporation
  • Which ever method used, the result is called
    transformation which is a change in the recipient
    cells genome caused by the acquired transgene.

30
Chemical Transfection Techniques
  • Calcium phosphate method involves the formation
    of a fine DNA/calcium phosphate co-precipitate
    which first settles on the cells and then
    internalized by endocytosis.
  • The precipitate must be formed freshly at the
    time of transfection. The DNA escapes and reaches
    the nucleus and can be then expressed. Since the
    cells must be coated by the calcium complex,
    monolayers of cells must be used for maximum
    efficiency. However, this method gives only 1-2
    transfection efficiency.

31
  • Transfection with polyplexes this is more
    efficient than that with calcium as it gives more
    uniform particle size. Polyplexes are a
    polycationic compounds that form soluble
    complexes (polyplexes) through spontaneous
    electrostatic interaction with DNA.
  • This method is adapted as a plasmid DNA transfer.
    The efficiency of the method can be increased by
    exposing cells to osmotic shock or treatment of
    cells with chloroquine.
  • Anew generation of the polycationic compounds has
    been developed such as poly-L-lysine and
    synthetic poly amines, polyethelylenimines and
    dendrimers (highly complex molecules built in
    layers from a central initiator such as ammonia
    or ethylenediamine).

32
Transfection with liposomes and lipoplexes
  • This can be done by packaging the DNA inside a
    fusogenic phospholipid vesicle which interacts
    with the target cell membrane and facilitate DNA
    uptake.
  • Briefly, bacterial cells will be transformed with
    suitable plasmid vector and treated with
    chloramphenicol to amplify plasmid copy number.

33
  • The cells will be treated with lysozyme to remove
    cell wall, resulting in protoplasts that will be
    centrifuged gently onto a monolayer of mammalian
    cells to promote fusion among them using
    polyethylene glycol.
  • Using lyposomes is more commonly used for this
    type of transfection. This method is commonly
    known as lipofection. This method is far more
    efficient than chemical transfection method. With
    this method up to 90 of cells in culture dish
    can be transected.

34
Physical transfection methods
  • Electroporation
  • Ultrasound
  • both methods create transient pores in the cell

35
Electroporation
  • is a physical transfection technique involves
    creating transient nano-meter size pores in the
    cell membrane by exposing cells to a brief pulse
    of electricity. The most critical parameter is
    the intensity and duration of electrical pulse.
  • Electroporation can be used for in vivo gene
    transfer particularly for surface or near surface
    tissue such as skin, muscle and certain tumors or
    even internal tissues such as liver. This can be
    achieved by direct application of electrodes to
    the skin following shaving and mild abrasion with
    the DNA being injected into the skin before
    electroporation.

36
Ultrasound transfection
  • Involves the exposure of cells to a rapidly
    oscillating probe such as the tip of sonicator.
    In this method the application of ultrasound
    waves to a dish or cells or a particular tissue
    results in the formation and collapse of bubbles
    in the liquid, including the cell membrane, a
    process known as cavitation.
  • The transient appearance of such cavities allows
    the DNA to cross the membrane into the cytoplasm.
    This method can be used both for in vivo or in
    vitro as the plasmid DNA is left structurally
    intact. As for electroporation, the DNA will be
    injected and then the ultrasound will be applied.

37
DNA microinjection
  • Direct transfer of DNA into the cell without a
    carrier is called DNA microinjectin. This can
    only be done for only few cells at a time. This
    technique is used mainly for large cells such as
    oocytes, eggs and the cells of early embryos. The
    DNA can be directly injected into tissues, such
    as skin, muscle or internal organs or it can be
    injected into the blood.

38
  • The process is remarkably efficient. Up to 60-
    66 of the embryos survive injection and up to
    25-30 of the embryos transferred to the oviduct
    survive to birth and about 25 of pups are
    transgenic (transgenic founders). Thus, from 1000
    inoculated fertile eggs, 30-50 (3-5) transgenic
    pups are produced.
  • The injected DNA gets incorporated at random
    sites within the genome and often multiple copies
    are incorporated at one site, therefore, not all
    the transgenic animals will have the desired
    traits.

38
39
Particle bombardment
  • is another direct delivery method initially
    developed for the transformation of plants. This
    method involves coating small metal particles
    with DNA and accelerating them into target
    tissues using a powerful force such as the blast
    of high pressure gas or an electric discharge
    through a water droplet. In animals, this method
    is used for tissues such as skin cells in vivo
    rather than cultured cells.

40
Bacterial vectors for gene transfer
  • The exploitation of living bacteria for gene
    transfer is central to the genetic manipulation
    of plants. Agrobacterium tumefaciens and its
    close relatives have been used for 20 years to
    generate transgenic plants.
  • Recently, A. tumefaciens has been used to
    transfer DNA to human cells. The protoplast
    fusion technique can be considered as highly
    efficient form of bactofection. However, this
    type needs human intervention, while the A.
    tumefaciens does not require human intervention.

41
How does the bacterial transfer of DNA happen?
  • The bacteria invades the host animal cells and
    undergo lysis within them releasing their plasmid
    DNA. Example of these bacteria including
    Salmonella species (lysis occurs in the
    phagocytic vesicle), Listeria monocytogenes and
    Shigella flexneri (lysis occurs for these two
    species after they escape from the vesicle).

42
  • The plasmid DNA then finds its way to the nucleus
    where it gets incorporated with the cells genome
    and gets expressed.
  • Contrary to the above mentioned bacteria, A.
    tumefaciens does not invade the cell, instead, it
    attaches itself to the outside surface followed
    by conjugation.

43
Viruses That are used as gene transfer vectors
  • Virus particles have a natural ability to adsorb
    to the surface of the cells and gain entry. This
    can be exploited to deliver recombinant DNA into
    animal cells.
  • Several classes of viruses has been used for gene
    therapy and at least 8 has been used in clinical
    trials. Transgenes may be incorporated into viral
    vectors either by addition to the whole genome or
    by replacing one or more viral genes. This can be
    done by ligation or by homologous recombination.

44
  • If the transgene replaces a none essential gene
    the vector is described as helper-independent
  • If it replaces an indispensable gene, then this
    vector will be helper dependent.
  • It is generally recommended to use vectors from
    which all viral coding sequences has been deleted
    such vectors are described as fully deleted or
    gutted or gutless vectors. These vectors contain
    just the cis-acting elements required for
    packaging and for genome replication.

45
Advantages of such vectors
  • high capacity for foreign DNA
  • because no viral gene products are made, the
    vector has no intrinsic cytotoxic effects.
  • Adeno virus
  • These are viruses with a linear, double stranded
    genome, of approximately 36 kb. These are used
    frequently due to certain advantages
  • stability
  • high capacity for foreign DNA
  • wide host range including none-dividing cells
  • Ability to produce high titer stocks up to 1011
    pfu/ml.

46
Adeno-associate virus (AAV)
  • These viruses are not genetically related to
    adenovirus but is so-called because it was first
    discovered as a contaminant in an adenovirus
    isolate.
  • The AAV is a single stranded DNA and is a member
    of the parvovirus family.
  • It is naturally replicating deficient, thus it
    requires the presence of another virus such as
    adenovirus to complete its infection cycle. AAV
    replicates lytically and produces thousands of
    progeny virions.

47
  • The dependence of AAV on a heterologous helper
    virus such as adeno virus provides an unusual
    degree of control over vector replication making
    AAV one of the safest vectors to use for gene
    therapy.
  • Other advantages of this viral vector is the wide
    host range that it exhibits including none
    dividing cells.

48
  • The AAV genome is small (5 kb) and
  • comprises a central region containing rep
    (replicase) and cap (capsid) genes flanked by
    145 kb inverted terminal repeats.
  • Foreign DNA replaces the cap region and gets
    expressed by indigenous promoter. However rep
    proteins might interfere with the expression
    process thus responsible for some of the
    cytotoxic effects of the virus.

49
  • New vectors are designed with the deletion of the
    rep and cap genes and only utilizing the
    repetitive sequence which are the only elements
    required for replication, transcription and
    proviral integration.
  • AAV vectors have been used to introduce genes
    efficiently into many cells including liver,
    muscle, and neurons.

50
Baculovirus vectors (BV)
  • Baculovirus promote high level of transgene
    expression in insect cells but can also infect
    mammalian cells.
  • Have rode shape capsid and large double-stranded
    DNA genomes. They productively infect arthropods
    particularly insects.
  • BV vectors are used mainly for highlevel
    transient protein expression in insects and
    insect cells.

51
  • These viruses produce a protein called polyhedron
    at a very high expression level yet this protein
    is not necessary for the infection process
    therefore it can be replaced with foreign DNA
    that can be expressed at high levels (up to 1 mg
    / 106 cells) under the control of the
    endogenous polyhedron promoter.
  • The highest amount of expressed proteins was
    initially achieved when the transgene was
    expressed as a fusion protein by incorporating it
    with the first 30 amino acids of the polyhedron
    protein.

52
  • There are two types of BV that are used as
    vectors
  • Autographa californica multiple nuclear
    polyhedrosis virus (AcMNPV) infects mainly
    insects
  • Bombyx mori nuclear polyhedrosis virus (BmNPV)
    infects insects silkworms and is used for
    production of recombinant proteins in silk worm
    larvae.

53
  • One problem of expression of mammalian proteins
    in insects is the different types of
    glycosylation. Thus as a solution, is to exploit
    the indefinte capacity of BV vectors to
    co-express multiple transgenes and thus modify
    the glycosylation process in the host by
    expressing appropriate glycosylation enzymes
    along with the transgene of interest.
  • Example this strategy was used to co-express fowl
    plague hemagglutinin and beta-1,2-N-acetylglucosam
    inyl-transferase resulting in the synthesis of
    large amounts of hemagglutinin correctly modified
    with N-acetylglucosamine residues.

54
  • The construction of baculovirus expression
    vectors involves inserting the transgene
    downstream of the polyhedron promoter. This is
    normally achieved by homologous recombination
    using plasmid vector carrying a baculovirus
    homology region.
  • Baculoviruses can also be used for the delivery
    of foreign genes to viruses to produce a cell
    model or animal models for certain diseases.
    Example the hepatitis C virus does not infect
    cultured cells, but a hybrid baculovirus
    containing the entire HCV genome can initiate an
    HCV infection.

55
  • Genotyping Transgenic Mice by PCR to Screen for
    Potential Founders
  • This is the test method of mice for the presence
    of the transgene by PCR.
  • Since we know the sequence of the gene that was
    inserted into the male pronucleus, we could
    determine if the mouse contains the transgene of
    interest, by performing PCR.
  • Tail biopsies from potentially transgenic mice
    will be obtained 5 weeks after injecting eggs (3
    weeks gestation time and 2 weeks of post-natal
    growth). The investigator then extracts DNA from
    the tail tips and tests for the transgenic by
    PCR. How?

55
56
  • By designing a set of primers that are taken from
    the transgene sequence and using them in a
    regular PCR to amplify the gene of interest if
    found.
  • Now if the mouse is a transgenic mouse, then a
    PCR product corresponding to a known size will
    appear in the gel. But if the mouse is NOT a
    transgenic one, there should be NO PCR product
    corresponding to that size.
  • In addition, to evaluate the stability of the
    insert, some markers has to be checked and the
    best ones are the ones that can be assayed
    readily such as observing the new phenotype of
    the progeny.

57
  • To determine the number of copies and places,
    Southern Blotting Analysis will be done
  • When pups are 6 weeks old, Southern blot analysis
    should be done to determine how many copies of
    the transgene were integrated, how many
    chromosomal sites the transgene inserted into, to
    verify transgenic status and to determine if the
    transgene is intact.
  • With this information, transgenic founders with a
    good chance of transmission (at least 5-10
    copies) of an intact transgene in a single
    insertion site can be selected for intensive
    breeding. (Figure 1 ).

58

59
  • One of the problems is that when DNA is
    micro-inserted, randomly some parts of it will
    replace some genes in the mouse, and thus might
    inactivate them.
  • Depends on which gene is inactivated, a damage to
    the progeny might occur.

60
  • Engineered Embryonic Stem Cell Method
  • In this method, cells from the Inner Cell Mass
    (ICM) of early embryos blastocysts (a stage of a
    developing mouse embryo) will be used.
  • These cells can be grown in cell culture and
    still retain the capability of differentiating
    into other cell types including germ line cells
    after they are introduced into another blastocyst
    embryo.
  • Such cells are called pluripotent (multi)
    embryonic stem (ES) cells. These cells can be
    easily manipulated by genetic engineering without
    changing their pluripotency.

61
  • Steps of the procedure
  • 1. Obtain fertilized eggs (pre-implantation
    zygotes) from a pregnant mother mouse as
    described above.
  • 2. Grow zygotes in culture until day 3.
  • 3. Harvest the Inner Cell Mass (ICM) from 3 day
    old blastocysts.
  • 4. Culture the Inner Cell Mass (ICM) on feeder
    cells to develop Embryonic Stem (ES) Cell lines.

62
  • 5. Create transgenic ES cells by microinjection
    or by introducing cells briefly to an electrical
    potential that disrupts cell membrane thus allows
    the entrance of DNA containing the transgene
    that was constructed with the genes of
    interest.
  • In this method a functional transgene can be
    integrated in the place of a dispensable gene in
    the genome of the ES cell.

63
  • 6. Inject the transgenic ES cells into the
    blastocoele (fluid filled cavity of the mass of
    cells) of a new 3-day old host blastocyst.
  • The injected ES cells combine with the host ICM
    and contribute to the developing embryo.
  • The first generation offspring are chimaeras -
    they have somatic cells composed of both
    transgenic ES cells and host cells
  • And also have germ cells composed of both
    transgenic ES cells and host cells
  • Usually a coat color gene is used in the
    transgene construct as a visual marker to
    facilitate the quick detection of the transgenic
    (chimaeric) pups.

64
  • 7. The transgene, if in the germ cell lineage,
    can be transmitted to offspring and homozygous
    transgenic lines can be constructed.
  • After transfection of ES cells in culture with
    the DNA vector
  • Some cells will have DNA integrated at
    none-target (spurious) sites
  • Some cells will have DNA integrated at target
    (correct) sites
  • Some cells will not have any DNA integration

65
  • How to enrich DNA integration at the specific
    sites?
  • A procedure called positive/negative selection
    is implemented.
  • This procedure used positive selection for cells
    did not accept the DNA inserts and negative
    selection for cells who have DNA integrated any
    where in their genome.
  • In this procedure, a construct will be prepared
    and should contain the following
  • Two blocks of DNA sequences (HB1 and HB2) that
    are homologous to separate regions of the target
    site.

66
  • The trans gene, TG that will confer a new
    function on the recipient
  • Neor, a DNA sequence that codes for an enzyme
    that inactivates neomycine and its relatives such
    the drug G418 which is lethal to mammalian cells

67
  • Two different genes for the thymidine kinase
    (tk1 and tk2). These enzymes phosphorylates the
    nucleoside analogue called gancyclovir.
  • DNA polymerase fails to discriminate
    against the resulting nucleotide and inserts this
    nonfunctional nucleotide into freshly-replicating
    DNA. So gancyclovir kills cells that contain the
    tk gene.
  • Now the arrangement of these sequences is key to
    the positive and negative selection procedure.

68
  • Possible results
  • Most cells fail to take up the vector these
    cells will be killed if exposed to G418 as the
    neo gene will not be incorporated. (positive
    selection).
  • In a few cells the vector is inserted randomly
    in the genome. In random insertion, the entire
    vector, including the tk genes, is inserted into
    host DNA. These cells are resistant to G418 but
    killed by gancyclovir. (Negative selection).
  • In still fewer cells homologous recombination
    by double crossover at target sites occurs. i.e
    Stretches of DNA sequence in the vector find the
    homologous sequences in the host genome and the
    region between these homologous sequences
    replaces the equivalent region in the host DNA.
  • Therefore, tk genes will be excluded and cells
    survive both G418 and gancylovir as only the neo
    and the trans genes are included.

69

70
  • Now by this method ES cells that carry the target
    site
  • will be enriched several thousand fold, thus
    better chances of producing a transgenic animal
    with the desirable characters.
  • By this method, ES cells that contain the target,
    are identified and cultured for propagation.
  • Embryonic stem cells carrying an integrated
    transgene can be cultured and inserted into
    blastocyst stage embryo and these embryos can
    then be implanted in pseudopregnant foster
    mother.

71
  • Transgenic lines can then be established by first
    mating founder transgenic mouse to animals from
    the same strain and then crossing transgenic
    litter mates to create a homozygous transgenic
    animal.
  • Unfortunately pluripotent ES cells comparable to
    those of mouse were not found in cattle, sheep,
    pigs or chickens.

72
  • Scientific and medical applications of the ES
    cells method of transfection
  • This route has been usually employed to
    inactivate a gene, alter it, or replace its
    protein coding region with a reporter (a coding
    unit whose product is easily assayed. It may be
    connected to any promoter of interest so that
    expression of the gene can be used to assay
    promoter function).
  • Main application of ES cell transgenic mice are
    to medicine and pure science including
  • Improve understanding of all aspects of healthy
    animal
  • Understanding therapeutic approaches
  • Understanding biochemistry and physiology
    particularly mammalian development,
    neurobiology, learning and memory.

73
  • Nuclear Transfer Method (non-transgenic method)
  • In this process the sheep Dolly was generated
    from an enucleated (nucleus was removed) egg into
    which the nucleus from a cultured somatic cell
    of a mature sheep has been introduced.
  • Method
  • Oocytes are recovered from animals between 28-33
    hours after injection of gonadotropin releasing
    hormone,
  • Oocytes are recovered in PBS containing 1 FCS
    and transferred to a new media containing 10 FCS
    and incubated at 37 C.
  • Nucleus is removed manually from an unfertilized
    oocyte as soon as possible

74
  • The somatic cell has to be in a non-dividing
    stage (G0) why ? this can be done in culture by
    depriving it of external stimuli that provokes
    growth. How?. Read the provided article
  • A non-dividing somatic cell is placed in contact
    with the oocyte and the two are fused together by
    applying an electrical potential which also
    activates the egg thus, mimicking the process of
    natural fertilization.
  • The result of this hybridization is an activated
    oocyte with two chromosome sets (from the diploid
    somatic cell).

75
  • Usually, the cytoplasm of normal oocyte contains
    proteins and RNA molecules that are required for
    the early stages of development but in this case,
    the cytoplasm of the somatic cell contains a
    whole set of genes that are reprogrammed to take
    control over the developmental program in the
    same way as the genes of the normal embryo.
  • Effect of age it was found that cells obtained
    from fetuses and new borne donors are more
    efficient in nuclear transfer while clones
    derived from adult cells show more abnormalities.
  • Why? It could be due to the fact that somatic
    cells of adult animals have accumulated more
    mutations or they are more differentiated than
    fetal cells thus are more likely to fail the full
    term development.

76
  • Applications of Nuclear Transfer
  • Nuclear transfer has applications outside the
    field of transgenesis such as propagation of an
    animal with a particularly desirable set of
    genes.
  • Since propagating the transgenic animals are not
    easy and normally goes with risks, the nuclear
    transfer can therefore, be used for propagating a
    successful transgenic animal making a whole herd
    of that animal !!! .. Prohibitively expensive.

77
  • Applications of transgenic animals
  • Transgenic mice
  • Transgenic mice can be used for
  • As test subjects to determine the effectiveness
    of potential therapeutic agents
  • Although mice are far from humans, some times
    they can serve as models for human diseases.

78
  • Specific Applications of Transgenic Mice
  • Transgenic Mice in Oncology
  • The study of transferred oncogenes has always
    been hampered by the fact that cell lines in
    culture have already been transformed to an
    abnormal phenotype.
  • The ability to insert oncogenes or
    proto-oncogenes into embryos and to study their
    effects in normally differentiating cells of an
    intact organism has circumvented this problem.
    Results of such studies have made an enormous
    contribution to our understanding of neoplastic
    diseases and its relationship to aberrant gene
    expression.

79
  • Transgenic Mice as Animal Models of Human
    Diseases
  • Animal models for human illnesses are useful for
    studying the pathogenesis of diseases as well as
    for developing and testing new therapies. Human
    diseases can be induced in transgenic mice by
    expression of transferred genes, or by
    insertional disruption of endogenous sequences.
  • Some examples of models created by transgene
    expression are listed below.
  • Hepatitis B is a human disease that lacks a
    readily workable animal model. Introduction of
    the HBSAg gene into mice results in transgenic
    mice that mimic the carrier state with production
    of HBsAg in the liver but with an absence of
    disease

80
  • Transgenic Mice as Models for Gene Therapy
  • Genes can be inserted into transgenic animals and
    function to alleviate disease states, such model
    systems can be of great importance in improving
    our understanding of the potential for gene
    transfer as an approach to treatment of diseases.
  • Mice with growth hormone deficiency are markedly
    reduced in size and males suffer from
    infertility. Introduction of the growth-hormone
    gene into these animals leads to growth which
    exceeds that of normal animals and restores male
    fertility.
  • However, the pattern of release of growth hormone
    that results from transgene function is
    apparently inconsistent with female fertility.
    i.e does not restore it.

81

82
  • Another Example
  • Insertion of either the mouse or human ß-globin
    gene can reduce the severity of ß-thalassemia in
    mice. In these experiments, the product of the
    human globin gene was able to associate
    effectively with the mouse A chains, and it
    actually functioned better than the transferred
    mouse gene ß-globin in reducing severity the
    thalassemic state.
  • Mice with a deficiency in gonadotropin-releasing
    hormone (GnRH) are infertile and exhibit profound
    perturbations of their reproductive endocrine
    functions. Cloning of the GnRH gene and its
    transfer into mice has resulted in restoration of
    normal endocrine function and in fertility.

83
  • Alzheimers disease model
  • Alzheimers disease is a degenerative brain
    disorder that is characterized by the progressive
    loss of both abstract thinking and is accompanied
    by personality change, language disturbance and
    a slowing of physical capabilities.
  • The brain of those patients accumulate within the
    body of the neurons a dense material called
    senile plaques.
  • The principal component of senile plaques and
    amyloid bodies is a 4-kDa protein called ßA4 (or
    ß protein). This protein is the product of an
    internal proteolytic cleavage of the ß- amy1oid
    precursor (APP).
  • Researchers have found that some strains of mice
    produce senile plaques during their life span,
    whereas others do not. Thus, the later (none
    producers) strains are important for forming
    transgenic mice that carry and express a
    transgene encoding the ßA4 portion of APP which
    might provide a model for studying the molecular
    basis of Alzheimers disease.

84
  • Importance of such experiment this type of mice
    can be used for a precise determination of the
    mechanism of Alzheimers disease and probably for
    a treatment scheme.
  • One of the vectors that has been constructed for
    modeling Alzheimers disease in mice consists of
  • A promoter region from brain specific virus
    ligated to a portion of the human APP (ß amyloid
    precursor protein) gene that encodes the last 100
    amino acids at the C terminus of APP, which
    includes the ßA4 amino acid sequence.
  • Transgenic mice were established with this
    construct, and expression of the transgene was
    confined to neurons of the brain.
  • Immunocytochemical studies showed that the brain
    of transgenic mice accumulated ßA4 protein that
    was derived from the transgene. How?

85
  • This screening can be done using anti- ßA4
    antibody that is conjugated to a dye which makes
    it visible either to the naked eye or under
    special microscope such as fluorescence.
  • Alternatively, ES cells that have a site-directed
    mutated APP gene could be used to establish a
    transgenic line that might mimic Alzheimers
    disease more precisely.
  • Transgenic mice have also been used as models for
    expression systems that are designed for
    secretion of the product of a transgene into
    milk.

86
  • Another example (CF)
  • Another example of the usefulness of the
    transgenic mice is the production of large
    quantities of authentic cystic fibrosis
    trans-membrane regulator (CFTR) that are needed
    to study its function and possibly formulate
    potential therapies for treating cystic fibrosis.
  • CFTR normally acts as a chloride channel but when
    its function gets altered, cystic fibrosis occurs
    and it will be characterized by the accumulation
    of mucus into the lungs and pancreas.

87
  • What is Cystic fibrosis?
  • It is the most common lethal human hereditary
    disorder, occurring once in every 3,000 births.
  • It affects the lung, intestinal tract and liver,
    with thick mucus, chronic airway infections and
    inflammation beginning in early childhood and
    leading to progressive loss of lung function.
  • While the life expectancy of these children is
    double what it was, they are still only expected
    to live to 40. The underlying defect is in a gene
    that codes for a substance that regulates protein
    secretion across a cell membrane, but infection
    also plays a major role.
  • All existing therapies only alleviate the
    symptoms by reducing infection and mucus.

88
  • To get large amounts of CFTR
  • A full length CFTR cDNA sequence was cloned into
    the middle of a defective goat ß-casein gene
  • The construct retained the promoter and the
    termination sequences of the goat ß-casein gene.
  • The ß-casein gene is then actively expressed in
    mammary glands during lactation producing the
    ß-casein which is the most abundant protein in
    the milk.

89
  • Now, transgenic mouse lines carrying the CFTR
    sequence under the control of the ß-casein gene
    regulatory sequences were established.
  • The product is milk from transgenic females
    contained the CFTR protein bound to the membrane
    of fat globules.
  • This is a model, however, to obtain mega
    quantities of this protein, a construct has to be
    introduced into a larger animal such as sheep,
    cows or goat.

90
  • Antisense Genes in Transgenic Mice
  • Another method for negating gene function
    involves the use of antisense transcripts.
  • When genes are cloned in reverse orientation with
    respect to the promoter, RNA may be produced from
    the non-coding strand.
  • This RNA, presumably by forming a heteroduplex
    with the sense RNA, can block translation of
    cytoplasmic mRNA.
  • Thus, antisense genes can be used to obliterate
    (wipe out) production of proteins from specific
    genes in transgenic animals.
  • The feasibility of this approach has recently
    been demonstrated by the transfer of an antisense
    construct of the gene for myelin basic protein
    (MBP) into mice. Interference with the production
    of MBP resulted in dysmyelination. Although this
    research is still its infancy, it has great
    potential for future experiments.

91
  • Transgenic cattle
  • If the mammary gland is to be used as a
    bioreactor, then dairy cattle are the likely
    candidates for transgenesis as they produce about
    10,000 liters of milk/year with 35 gm
    protein/liter.

92
  • Protocol to produce transgenic cattle (Figure
    15-9)
  • collecting oocytes from slaughterhouse killed
    cows
  • in vitro maturation of these oocytes
  • in vitro fertilization with bull semen
  • centrifugation of fertilized eggs to concentrate
    the yolk so that male pronuclei will be seen
    under the dissecting microscope.
  • microinjection of input DNA into male pronuclei
  • in vitro development of embryos
  • embryo implantation into a recipient foster
    mother
  • DNA screening of the offspring for the presence
    of the transgene.
  • When this procedure was done only two transgenic
    calves were produced from a starting pool of 2470
    oocytes which means that the procedure is
    feasible but in efficient in this format.

93
  • Goals of producing transgenic cattle
  • To change the constituents of milk. For example
    the amount of cheese produced from milk is
    directly proportional to the amount of k-casein
    content of the milk so if a transgene is
    constructed to produce milk with higher amounts
    of k-casein, then the production of cheese will
    increase proportionally.
  • Production of transgenic cows with modified genes
    to produce lactose free milk could solve the
    problem of those who have lactose intolerance.
  • For livestock in general, attempts to produce
    animals with inherited resistance to bacterial,
    viral, and parasitic disease is a goal. Example
    of major diseases that affect the livestock are
    mastitis in cows, neonatal dysentery in swine,
    fowl cholera.
  • If the basis of each of these is a single gene
    that will be responsible for the resistance, then
    it might be possible to produce transgenic
    animals that carry this gene.

94
  • Other alternative, is the production a transgenic
    animal with inherited immunological protection.
  • A number of candidate genes that contribute to
    the immune system such as Major
    histocompatibility genes, T-cell receptor genes,
    lymphokine genes are under study to evaluate this
    potential.
  • But the most favorable preliminary results to
    date comes from research in which the genes
    encoding the heavy and light chains of a
    monoclonal antibody (MAb) have been transferred
    to mice, rabbits and pigs.

95
  • By this introduction of MAb, these animals will
    have an endogenous source of MAbs with predefined
    specificity toward certain pathogen, thus
    eliminates the need for immunization. This
    concept is called In vivo immunization.

96
  • Example the genes for the immunoglobulin chains
    of a mouse MAb that are specific to
    4-hydroxy-3-nitrophenylacetate were cloned in a
    tandem and microinjected into fertilized egg of
    mice, rabbits and pigs.
  • In each case MAb activity was found in the serum
    but the concentrations of the antibodies were low
    which could be due inheritable problems of the
    construct, thus a new construct should be tested.

97
  • Transgenic Sheep
  • Transgenesis research with sheep, goat or pigs
    has concentrated in the most part on utilizing
    their mammary glands as bioreactors for
    production of pharmaceutical proteins.
  • Example Production of transgenic sheep that
    produces anti-trypsin in their milk This protein
    is a potential treatment for cystic fibrosis.
  • The Technology
  • PPL Therapeutics transfers genetic material from
    one organism to another using the same technology
    it used to produce "Dolly the Sheep", a process
    called somatic cell nuclear transfer.

98
  • Steps of the procedure
  • Genes are Modified A single cell from a sheep is
    modified to include the human gene for the
    protein alpha-1 antitrypsin. However, the gene
    must only turn on in the mammary glands so that
    the protein only appears in the sheep's milk.
  • Before the sheep DNA is modified, the human gene
    is fused to the promoter gene for
    beta-lactoglobulin. The human gene will only be
    expressed when the beta-lactoglobulin is turned
    on, and this only happens in the milk-producing
    mammary glands.
  • Injection The nucleus, containing the modified
    DNA, is removed from this cell and injected into
    the enucleated fertilized sheep oocyte. Or the
    modified somatic cell could be fused to the
    enucleated oocyte.

99
  • Implantation of embryos The fertilized sheep
    embryo is implanted into a surrogate mother for
    the rest of its pregnancy.
  • Lactation Upon giving birth to a lamb the
    mothers (ewes) produce milk (lactated). Beta
    lactoglobulin production started during
    lactation, so did production of human alpha-1
    antitrypsin. The rams also contain the required
    gene but it is not active, although it can be
    passed to their offspring. The alpha-1
    antitrypsin protein that is expressed in the milk
    can be extracted and purified.
  • Next Generation The newborn lambs were screened
    for presence of the gene (by DNA analysis of tail
    tissue or blood from the jugular) and mated when
    mature.
  • The production flock was started from semen from
    two transgenic rams brought to New Zealand in
    1996. Conventional New Zealand ewes were
    inseminated and some of the resulting lambs were
    transgenic. Embryos from transgenic animals were
    transferred to surrogate mothers.

100
  • The modified gene is shown to be stable (i.e has
    been transmitted faithfully from parent to
    offspring). How can we judge that?
  • Homozygotes are as healthy as heterozygotes this
    shows that the gene has not inserted into an
    essential part of their genetic material -
    insertion into other parts of the DNA would lead
    to death of the offspring
  • The human protein secreted in the milk has been
    consistent in quantity and quality

101
  • The Benefits
  • The most obvious benefit from this research is
  • Production of a treatment for cystic fibrosis.
    How? See next slide

102
  • Human alpha-1 antitrypsin is currently derived
    from blood plasma and administered intravenously
    at 60mg/kg once a week. The difficulties with
    this treatment are
  • 1. Cost - treatment for an individual costs
    40,000 per year
  • 2. Availability - the protein is produced in
    plasma at a concentration of about 1.5 g/L and
    obtained from healthy donors.
  • 3. Contamination - any extraction of material
    from blood carries risk of contamination from
    other diseases such as HIV, new variant
    Creuzfeldt-Jakob Disease (BSE) and Hepatitis B.
  • 4. Efficiency - using transgenic animals produces
    far greater quantities of the protein at lower
    cost in the long term, this research will also
    provide further benefits
  • Provide techniques for producing other
    disease-fighting drugs
  • Provide techniques for incorporating medicines
    in foods
  • Help scientists to understand how milk protein
    is produced and modified
  • Transgenic-derived proteins were glycosylated
    and had biological activities comparable to those
    extracted from human sources.

103
  • Goats and Pigs
  • Generally the production of transgenic goats and
    pigs is similar to that for sheep however, there
    are some differences in that the
  • expression of transgenes in the mammary glands of
    sheep or goats had no ill effects on either
    lactating female or nursing progeny.
  • While the transgene for bovine growth
    hormone-under the control of the metallothionine
    promoter- when introduced into pigs, several
    adverse results were observed
  • Gastric ulceration
  • Kidney dysfunction
  • Lameness
  • Inflammation of the lining of the heart
  • Swelling of the joints
  • Susceptibility to pneumonia

104
  • Transgenic Birds
  • Avian ova are normally fertilized approximately
    30 minutes after ovulation. Cell division occurs
    in the oviduct for approximately 20 hours before
    ovi position. At this time, the embryo is
    comprised of approximately 60,000 pluripotent
    cells, which are collectively called the
    blastoderm.
  • The presence of a large yolk and multiple
    pronuclei makes direct microinjection of DNA
    impractical.
  • Therefore, DNA microinjection into fertilized
    bird eggs to produce transgenic strains is not
    possible

105
  • During fertilization in birds several sperms can
    penetrate the ovum, instead of only one as in
    case of mammals.
  • Therefore, it is not possible to identify the
    male pronucleus that will fuse with the female
    pronucleus.
  • Microinjection of DNA into cytoplasm is not
    enough for the process to proceed as the DNA will
    not integrate into the genome of the fertilized
    egg.
  • The technique also would be difficult as the
    avian ovum after fertilization become enveloped
    in tough membrane and surrounded by large
    quantities of albumin and enclosed in inner and
    out shell membranes.

106
  • By the time the avian egg outer shell membrane
    hardened, the developing embryo (blastoderm
    stage) will be two layers of 40,000 to 80,000
    cells.
  • At the moment no one has identified avian
    specific embryonic stem cells so this approach
    can not be used in birds. The alternative is a
    procedure using engineered cells from embryos.
    How??

107
Procedure Plastoderm cells are removed from the
donor chicken These cells get transfected with
cationic lipid (liposome) transgene DNA complexes
(lipofection). The cells will be reintroduced
into the subgerminal space of embryos of freshly
laid eggs. Figure 15-10 shows a schematic diagram
of this procedure. Some of the progeny will
consist of a mixture of cells with some cells
from the donor but most from the recipient, such
mixture is called the chimera Lipofection
delivery into eukaryotic cells of DNA and RNA or
other compounds that have been encapsulated in an
artificial phospholipid vesicle.

108

Now in some
of these chimeras cells that were descended from
transfected cells may become part of the germ
line tissue and form germ cells. Transgenic lines
can then be established by rounds of mating. The
proportion of chimeras can be increased to
enhance the probability of obtaining germ line
chimeras if the receiving embryos are irradiated
with a dose of 540-660 rads for 1 h prior to the
introduction of transfected cells. Irradiation
destroys some of the blasoderm cells thus
increasing the final ratio of the transfected
cells to the recipient cells.

109


What can we use transgenic chicken for? To
improve the genetic makeup of the existing
strains with respect to resistance to avian
viral and coccidial diseases, better feed
efficiency, lower fat and cholesterol in eggs
and better meat quality. The egg with its high
protein content could be used as a source of
pharmaceutical proteins

110
Transgenic Fish As natural fisheries become
exhausted, production of this source will depend
more on the aquaculture. Production of transgenic
fish therefore become a primary objective. To
date, transgenes have been introduced by DNA
microinjection into the fertilized eggs of number
of fish species including Catfish , Crap, Trout
, Salmon , Tilapia In fish the pronuclei are not
readily seen under the microscope after
fertilization, therefore, a linearized transgene
DNA is microinjected into the cytoplasm of either
fertilized eggs or embryos that have reached the
4 cell stage.

111
Now because fish eggs develop externally there
will be no need for implantation. Instead the
development can be done in the Temperature
regulated tanks with a survival rate from 35-80
and production of the transgenic fish ranges from
10-70. Same as in transgenic animals, the
founder fish can be mated and transgenic lines
established. In one study, a transgene
consisting of the promoter region of the
antifreeze protein gene of the fish called ocean
pout. The growth hormone cDNA from salmon. And
the termination polyadenylation signals from the
3 end of the end of the antifreeze protein. This
construct was injected into eggs of Atlantic
salmon.

112
Result the transgenic salmon was larger and grow
faster than the none transgenic. Eventually,
genes for disease resistant, tolerance to
environmental stress, and other biological
features will be introduced into fish in cold and
warm waters.

113
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