Chapter 21
1. How are transgenic mice created?
Transgenic mice can be produced by retroviral vectors that infect the cells of an early-stage
embryo prior to implantation into a receptive female. DNA may also be introduced into mice by
microinjection into the enlarged sperm nucleus of a fertilized egg. Finally, transgenic mice may
be produced by the introduction of genetically engineered embryonic stem cells into an earlystage developing embryo before implantation into a receptive female.
2. What is positive-negative selection, and how does it work?
. In case of the method where we engineer embryonic stem cells for inserting the target gene,
there are three things that can happen : i) the DNA may not get integrated in the cell genome at
all, ii) the transgene can get integrated randomly, iii) the gene could find its normal counterpart
and swap places by homologous integration. Therefore, to find out which of the three
possibilities has occurred we have to perform a selection called the positive negative selection.
The targeting DNA vector contains i) two blocks of DNA sequences homologous to separate
regions of the target site , ii) the transgene, iii) DNA sequence coding for neomycin resistance,
iv) gene for thymidine kinase from HSV which is outside homology and doesn’t recombine. For
the procedure, after insertion into ES cells, we grow the cells on neomycin; this kills any of the
untransformed cells (positive selection). Now, we treat the cells with ganciclovir. If the gene for
thymidine kinase is present, it produces toxin in presence of ganciclovir and kills the cells. So,
now the cells where the transgene has integrated randomly doesn’t survive because these cells
are resistant to neomycin but sensitive to ganciclovir (negative selection). Finally, we get the ES
cells with targeted disruption of the transgene.
3. What are knockout mice? How and why are they established?
A knockout mouse is a genetically engineered mouse where a gene for a particular function has
been disrupted or knocked out (removed) by inserting another gene to disrupt the function.
They can be established by using a selectable marker gene with flanking DNA sequences that are
homologous to regions of the targeted gene. Homologous recombination disrupts or knocks out
the targeted gene which completely ceases the function of the gene.
This process is important because it helps us in determining the particular function or the
developmental and physiological consequences of knocking out that particular gene which helps
us in identifying various abnormalities.
4. What are knockdown mice? How are they generated?
Knockdown mice are produced for similar reasons as knockout mice; in order to deduce the
purpose of a given gene. The knockdown approach downregulates (but does not abolish)
expression of a gene by preventing translation by RNAi. Exogenous dsRNA is introduced that
once incorporated into the RISC complex will seek out and destroy mRNA for the gene of
interest. Knockout and knockdown mice can also be used to study the pathology of human
diseases and to test possible therapies.
5. Describe how the Cre-loxP combination system is used to regulate the expression of a
transgene.
The Cre-loxP recombination system uses a line of transgenic mice that produce the cre gene
under the control of a cell-specific promoter. A loxP site is then cloned on each side of the exon.
Homologous recombination is used to introduce the construct into embryonic stem cells. The
transgenic lines are crossed and in cells that get both constructs the Cre recombinase gene is
active and two Cre molecules bind to the loxP sites, which undergo recombination leading to
excision of the exon between the loxP sites. Without the exon the remaining gene is inactive.
This system can similarly be used to activate a given transgene by removing a transcriptional
inhibiting sequence to activate expression of the gene.
6. What are the advantages and disadvantages of using transgenic mice as model systems
for human disease?
Transgenic mice are useful for studying human disease because they are also mammals, so it is
likely that diseases that affect humans will affect mice in a similar way. Both the onset and the
progression of the disease can be studied within this model. However, mice are not humans and
there are several important differences that still limit the applicability of mice model information.
Mice are a good place to start because it is likely that if a given therapy does not work in mice
that it will not work in humans however, if a system does work for mice there is still no
guarantee that the same therapy will work for humans.
8. What is nuclear cloning?
Nuclear cloning is the production of an organism by placing a nucleus from a somatic cell into
an enucleated fertilized egg. The fertilized egg is then implanted into a foster mother and will
grow into a full organism (clone of somatic cell). This process is also referred to as a nuclear
transfer. This method has been used to clone higher order mammals such as sheep, cattle, goats,
dogs, cats, and pigs.
10. Discuss how the mammary gland could be used as a bioreactor for the production of
commercial products.
The mammary gland could be used as a bioreactor for the production of commercial products
because annually 10,000 liters of milk are produced and scientists have found ways to use
mammary gland specific promoters in conjunction with genes to produce nutrients and vitamins
within the milk. The benefit is that the compounds can easily be separated or can be left in the
milk and directly consumed. For some proteins of which very little is needed, only a handful of
transgenic animals would be necessary to produce the entire world’s supply of the compound of
interest.
13. Why are pigs carrying a phytase transgene considered to be environmentally friendly?
Pigs that carry the phytase gene are considered environmentally friendly because they carry the
phytase gene in their salivary gland that can overcome the pollution consequences of phytase
being in the diet. The problem with feeding pigs a high phytase diet is that they cannot properly
break it down and as a result secrete large amounts of phosphorus in their excrement. The
solution is the inclusion of the phytase gene in the salivary glands so that the phytase is broken
down and less phosphorus is produced.
15. Discuss how transgenesis might improve fish aquaculture.
Fish aquaculture can be improved with transgenesis because it can lead to increased growth
rates, the production of fish that are tolerant to environmental stresses, and the production of fish
that are resistant to certain diseases. Transgenesis can also be used to monitor the amount of
certain aquatic pollutants by designing fish that can serve as biosensors for certain pollutants.