Animal biotechnology lecture 2

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Animal biotechnology lecture 2

• Dr. Ziad Jaradat

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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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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)

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• 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.

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• 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

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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Table of common vectors used for such purpose

• Vector Origin Insert
  size rangeMulticopy 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.

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• 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.

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• 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.

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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.

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• 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.

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• 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.

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oocyte
Pippet
Micro needle
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(No Transcript)
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• 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.

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• 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?

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• 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.

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• 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.

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• 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 ).

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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

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• 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.

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• 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

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• Nuclear Transfer Method (non-transgenic method)
• In this process the sheep Dolly is 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

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• 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).

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• 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.

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• 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.

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• 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.

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• 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.

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• 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

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• 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.

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• 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.

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• 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.

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• 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?

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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

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• 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 semin
• 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.

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• Goals of the 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.

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• 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.

66

• 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.

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• 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.

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• 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.

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• 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.

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• 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.

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• 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

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

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• 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.

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• 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

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• 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

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• 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.

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• 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??

78
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.

79

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.

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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

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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.

82
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.

83
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.

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• Thank you
• Do you think you learned some thing new?