Transgenic Animals:

1
Transgenic Animals The New Producers of Tomorrow
Melissa Glen, Novalene Naicker, Reyaska
Singh School of Biological and Conservation
Sciences , University of KwaZulu-Natal
Methods of Creating a Transgenic Animal
Transgenes A gene cannot be simply transferred
between animals in reality. It has to go through
a number of procedures first before inserting it
into the new host. The gene of interest obtained
from the donor animal is isolated and purified.
Next, the regulatory components are added to the
gene (Garvin et al., 1998). These components are
genetically engineered to target specific sites
in the host, as well as, to control the
expression of the gene i.e. to enhance or depress
certain characteristics by over- or
underexpressing the gene (http//www.wmin.ac.uk/r
edwayk/lectures/transgenic.htm).This transgene
is inserted into a vector and transformed into
bacteria to make many clones of this transgene
(Garvin et al., 1998). These clones are used to
increase the probability of the gene being
integrated into the host DNA.
Retroviral Vector Method (Hunter et al., 2005
http//www.clontech.com/support/tools.asp?product_
tool_id54271tool_id154907)
1. Donor female mouse from which the fertilized
oocyst is removed. The embryonic stem cells are
then removed from the inner cell wall of the
mouse blastocyst (early mouse embryo). These
embryonic stem cells are pluripotent (have the
ability to become any cell. (Picture Adapted
from www.nih.gov Wheeler, 2007)
1. The genes which code for proteins vital for
viral functioning, especially with regards to its
replication are removed. The cis ends of the
genome are left as these are important in
replication of the transgenic genome. The removed
genes are replaced with the transgene sequence
which should be of similar size to those genes
removed.
1. Virgin mice are treated with hormones to
synchronize their cycles so that super ovulation
occurs (i.e. produce a higher number of eggs than
normal). These eggs are harvested and fertilized
in vitro. Two pronuclei can be detected in the
eggs 8 to 12 hours following fertilization, each
containing genetic information from either the
mother or father.
2. A microtube is used to hold the fertilized egg
in place while an extremely fine glass needle is
used to inject a solution containing the
transgene (200-300 copies) into the male
pronuclei. Very few of the embryos survive this
process, and of these only a few successfully
incorperate the foreign DNA into their genome
4. The transformed embryonic cells (10-15) are
removed from culture and injected into
blastocysts (8-10) which were harvested from a
donor female. (Picture Adapted from Wheeler,
2007)
2. The new genome is replicated in vitro by
making the removed viral genes temporarily
available. The transgenic genome is then
recovered from the replication medium
Adapted from www.nih.gov
3. Packaging or helper cell lines are used to
package the transgenic genome as viral particles.
These viral particles are unable to replicate and
are thus no longer pathogenic
3. The pronuclei are then allowed to fuse
naturally to form a diploid zygote, which divides
by mitosis to form a 2-cell embryo. This embryo
is then transferred to the oviduct of a
pseudopregnant mouse.
5. These blastocysts are implanted (in vitro)
into the uterus of a pseudopregnant female mouse
(uterus is receptive to the developing embryo).
These embryos are left to develop within the
uterus of the female
Adapted from www.nih.gov
X
news.bbc.co.uk
For all methods pseudopregnant mice are used.
These are produced by mating a female mouse with
a sterile (neutered) male. This elicits the
hormonal changes needed to make her uterus
receptive to the implanted embryos
(http//www.wmin.ac.uk/redwayk/lectures/transgeni
c.htm). The chimeric offspring thus produced are
tested to determine whether any are transgenic
(Buy, 1997). This is done by removing some
tissue, usually from the tail of the offspring ,
isolating the DNA and analysing it to determine
the presence or absence of the transgene (Garvin
et al., 1998). These are then mated with a
non-transgenic mouse to produce heterozygous
offspring, which are then mated to produce mice
that are homozygous for the transgene (Garvin et
al., 1998). Continuous mating of such homozygous
Individuals will ensure the establishment of the
desired transgenic trait.
Adapted from Gilbert, 2004 (from biotech notes)
www.nih.gov
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• References
• Buy M (1997) Transgenic Animals.
  http//www.acs.ucalgary.ca/browder/transgenic.htm
  l (accessed on 29-09-2007)
• Donnelley S, McCarthy CR, Singleton R (Jr) (1994)
  The Brave New World of Animal Biotechnology. The
  Hastings Center Report 241 pp. S1-S31
• Garvin W, Harms U, Shearer C, Simonneaux L (1998)
  Transgenic Animals. Unit 11. European Initiative
  for Biotechnology Education
• Houdebine LM (2002) The methods to generate
  transgenic animals and to control transgene
  expression. Journal of Biotechnology 982 pp
  145-160
• Hunter CV, Tiley LS, Sang HM (2005) Developments
  in Transgenic Technology Applications for
  Medicine. Trends in Molecular Medicine 116 pp
  293 298
• Margawati E (2003) Transgenic Animals Their
  Benefits To Human Welfare. http//www.actionbiosci
  ence.org/biotech/margawati.html (accessed on
  29-09-2007)
• Wheeler MB (2007) Agricultural applications for
  transgenic livestock. Trends in Biotechnology
  255 pp 204-210

• http//www.askoxford.com (accessed on 04-10-2007)
• http//www.clontech.com/support/tools.asp?product_
  tool_id54271tool_id154907 (accessed on
  01-09-2007)
• http//www.copewithcytokines.de/cope.cgi?keytrans
  genic (accessed on 29-09-2007)
• Htttp//www.darwin.bio.uci.edu/tjf/tmftgms.html
  (accessed on 29-09-2007)
• http//www.psrast.org/mianunpr.htm (accessed on
  04-10-2007)
• http//en.wikipedia.org/wiki/Genetically_modified_
  organism (accessed on 04-10-2007)
• http//www.wmin.ac.uk/redwayk/lectures/transgenic
  .htm (accessed on 29-09-2007)

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