Using DNA Technology to Produce

Transgenic Animals
Transgenic Animals


Transgenic Animals: A Focus
on Transgenic Mice Studies


http://www.hku.hk/biochem/tgcentre/transcentre.html

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
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


Three different breeding pairs of mice are
required.
First Breeding Pair:
Fertile male + superovulated female
Fertile male = stud (changed regularly to ensure performance)
Superovulated female = immature female induced to
superovulate
Pregnant mares 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.


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.

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


http://www.itba.mi.cnr
.it/human_g...transgeni
c.html

IV. Manipulation of Fertilized
Oocytes
See Slides
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
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
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


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 (Pompes disease
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.