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Using DNA Technology to Produce Transgenic Animals. Sherry Fuller-Espie, Ph.D., DIC Associate Professor, Cabrini College © Sherry Fuller-Espie, 2003. Transgenic Animals . Transgenic Animals: A Focus on Transgenic Mice Studies http://www.hku.hk/biochem/tgcentre/transcentre.html.

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Using DNA Technology to Produce Transgenic Animals


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using dna technology to produce transgenic animals

Using DNA Technology to Produce Transgenic Animals

Sherry Fuller-Espie, Ph.D., DIC

Associate Professor, Cabrini College

© Sherry Fuller-Espie, 2003

slide3
Transgenic Animals: A Focus on Transgenic Mice Studieshttp://www.hku.hk/biochem/tgcentre/transcentre.html
i introduction
I. Introduction
  • Transgenic animals:
    • Animals which have been genetically engineered to contain one or more genes from an exogenous source.
    • Transgenes are integrated into the genome.
    • Transgenes can be transmitted through the germline to progeny.
    • First transgenic animal produced = “Founder Animal”
ii introduction of foreign genes into intact organisms
II. Introduction of foreign genes into intact organisms
  • Procedure is basically the same regardless of which animal is involved.
  • Integration usually occurs prior to DNA replication in the fertilized oocyte.
    • Majority of transgenic animals carry the gene in all of their cells, including the germ cells. Transmission to next generation requires germline integration.
    • Some integration events occur subsequent to DNA replication giving rise to mosaic animals which may or may not contain the transgene in its germline.
iii procedure for producing transgenic mice
III. Procedure for Producing Transgenic Mice
  • Three different breeding pairs of mice are required.
slide7
First Breeding Pair:
    • Fertile male + superovulated female
      • Fertile male = stud (changed regularly to ensure performance)
      • Superovulated female = immature female induced to superovulate
        • Pregnant mare’s serum (=FSH) on day 1
        • Human Chorionic Gonadotropin (=LH) on day 3
      • Mated on day 3
      • Fertilized oocytes microinjected on day 4 with foreign DNA construct.
      • Microinjected oocytes are transferred to the oviducts of surrogate mothers at end of day 4.
slide8
Second breeding pair:
    • Sterile male + surrogate mother
      • Sterile male produced through vasectomy
      • Surrogate mother must mate to be suitable recipient of injected eggs
      • Mated on day 3
      • Microinjected oocytes from first breeding pair are transferred to oviducts on day 4
      • Embryos implant in uterine wall and are born 19 days later.
      • Southern blotting techniques confirm presence and copy number of transgenes.
slide9
Third breeding pair:
    • Foster parents
      • Fertile male + female mated to give birth on same day surrogate mother
      • Serves as foster parent if caesarian section is required for surrogate mother
v gene expression in transgenic mice
V. Gene Expression in Transgenic Mice
  • In order to discriminate the products of the injected gene from those of an endogenous counterpart, the injected gene must be marked in some way.
    • Mini-genes where exons are deleted of cDNA where introns are absent.
    • Modification by insertion/deletion/mutagenesis of a few nucleotides (e.g. the gain or loss of a restriction endonuclease site).
    • Hybrid genes where foreign epitopes are expressed on transgenic products.
vi tissue specific gene expression
VI. Tissue-Specific Gene Expression
  • Generally, if a tissue-specific gene is expressed at all, then it is expressed appropriately, despite the fact that it has integrated at a different chromosomal location.
vii examples of studies utilizing transgenic mice
VII. Examples of Studies Utilizing Transgenic Mice
  • “Pharm” animals (transgenic livestock)
    • Bioreactors whose cells have been engineered to synthesize marketable proteins
    • DNA constructs contain desired gene and appropriate regulatory sequences (tissue-specific promoters)
    • More economical than producing desired proteins in cell culture
slide15
Naked human Hb from pigs

Human lactoferrin in cows’ milk

Alpha-1-antitrypsin in sheep

HGH in mouse urine (uroplakin promoters)

Human antibodies in mice (H and L chain tgenics  hybridomas)

CfTCR in goats

Tissue plasminogen activator (TPA) in goats

Human antithrombin III in goats

Malaria antigens in goats (vaccine)

Alpha-glucosidase in rabbits (Pompe’s disease

viii transgenic pigs for the production of organs for transplantation
VIII. Transgenic Pigs for the Production of Organs for Transplantation
  • Pig organs are rejected acutely due to the presence of human antibodies to pig antigens.
  • Once human antibodies are bound to pig organs, human complement is activated and triggers the complement cascade and organ destruction.
  • Transgenic pigs with complement inhibitors have been produced and are gaining feasibility as a source of xenogeneic organs for transplantation.