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CHAPTER 21
1. Transgenic mice can be created by cloning the transgene into a retrovirus and infecting
an early-stage embryo with the virus, by microinjection of DNA into the sperm
pronucleus of an embryo or by injecting transgenic embryonic stem cells into an earlystage embryo. The embryo is then implanted in the uterus of a surrogate mother, where, if
the DNA was incorporated into the embryo, a transgenic mouse develops. The retrovirus
method can be used only if the transgene is small (≈8 kb). A second disadvantage of this
system is that extra viral genomic DNA may also be incorporated into the host's
chromosome. On the other hand, lentiviral vectors are not limited to smaller transgenes
and can infect non-dividing cells. The microinjection technique is the one typically used
to create transgenic mice. A donor female is superovulated, mated and then killed to
harvest the eggs. The male pronucleus from the sperm is larger than the female
pronucleus and can be seen under a dissecting microscope. The linear DNA is then
injected into the male pronucleus. The disadvantage of this system is that it requires
highly trained technicians, and even then, only 5% of the eggs develop into transgenic
mice. The method is highly inefficient at each step and even if the DNA is integrated into
the embryo, there is a chance that due to positionality and copy number, the offspring
will still not be useful. The embryonic injection method utilizes genetically engineered
stem cells (which are easier to engineer than full embryos and eliminates the problem of
random integration of the transgene into the host chromosome). The chimeric mice are
then breed for a transgenic homozygote.
2. Positive/Negative selection works by a) killing off the cells that do not have vector
DNA anywhere in their genome, and b) killing off all of the transformed cells that did not
occur by homologous recombination and therefore do not have the transgene at the
desired location. The first case is considered positive selection and involves the insertion
of the gene for neomycin resistance. After the transfection of cells using the embryonic
stem cell method, the non-transformed cells are killed off by exposure to neomycin. Only
the transformed cells, with viral DNA somewhere in the chromosome are resistant to the
neomycin treatment. The second case is considered negative selection and is possible
because the thymidine kinase gene which is present in the injected DNA produces a lethal
toxin when treated with the drug ganciclovir. The thymidine kinase gene is present in the
vector DNA, but not within the “fake gene” region designed to mimic the site on the
chromosome where you want the transgene to insert. If the transfection occurred by
chromosomal rearrangement, then only the knockout gene fragment of the DNA
transferred to the chromosome, specifically knocking out the gene of interest and
carrying the neomycin resistance. If the transformation occurred by random insertion,
then the whole DNA insert, including the thymidine kinase gene, is present somewhere in
the chromosome, but not at the desired position.
3. Knockout mice are mice with a specific gene that is completely nonfunctional. Gene
expression has been abolished using the embryonic stem cell method of transgenesis
where a dysfunctional copy of the gene of interest replaces the functional gene through
homologous recombination. These lines are established to provided researchers with a
living organism without the functioning gene. By observing the mutant phenotype,
scientists are able to draw conclusions about the exact function of the gene in the wild
type animal.
4. Knockdown mice are mice where gene expression is decreased using RNAi. The
mouse has a mutant phenotype not because there is anything wrong with the wild type
gene, but because the RNA is eliminated preventing the production of protein.
Knockdown mice are made by cloning a portion of the gene of interest, followed by a
spacer, followed by the inverted portion of the gene of interest into a vector. The
sequence is arranged so that the RNA transcript will form a double-stranded hairpin. This
vector DNA is then added to the mouse embryo via microinjection or lentiviral vector.
RNAi (Dicer and RISC degrade the dsRNA and produce microRNAs which then bind to
the endogenous mRNA and mark them for degradation).
6. The advantages of using transgenic mice as a model system for human diseases are that
you can test out potential drugs and therapeutic agents on the mice before the treatments
ever go to humans. The etiology of complex diseases can also be examined in mice to
improve knowledge of how the disease functions and progresses. The main disadvantage
is that mice are not humans and there is no guarantee that a drug will work the same on a
mouse as it does on a human, or that a disease will affect mice the same as it does
humans (e.g. cystic fibrosis in humans generally manifests itself as a lung disease, but in
mice the dominating symptom is an intestinal phenotype).
7. A mouse system was developed for understanding Alzheimer disease in humans.
Alzheimer disease is a degenerative disorder of the brain where plaques develop at the
synaptic junctions of nerves and neurofibrillary tangles develop in the cell body. The
proteins produced by APP genes are the primary component of the senile plaques. Mouse
models were produced with altered APP genes. A brain-specific promoter was fused to
APP cDNA (with the introns added back). Overexpression of the PDAPP construct in
elderly mice produces the symptoms of Alzheimers (memory loss, neuron death and
senile plaques). The mice do not develop neurofibrillary tangles, though, so that is either
caused by a second gene or is specific to the human Alzheimer's phenotype. Transgenic
mice have also been produced with BACE1 knocked out. BACE1 is a protease that
cleaves APP. Transgenic mice that produce APP but do not have BACE1 and do not
develop senile plaques.
10. The mammary gland has been used as a bioreactor for the production of
pharmaceutical proteins. If transgenic animals are engineered with a mammary gland
promoter and the signal sequences for the secretory pathway, large quantities of a drug
can be purified out of the milk and sold for human consumption. The cystic fibrosis
transmembrane regulator (CFTR) protein, antithrombin III, growth hormone, and insulin
are just a few therapeutic proteins that are being expressed in transgenic animals.
Production of the protein in the mammary glands is particularly desirable since it
simplifies the collection and purification of the target protein.
13. Enviropigs are pigs that have been genetically engineered to carry a bacterial phytase
gene that allows for the digestion of phytate. The primary storage form of phosphorous
used by plants is phytate, but only ruminants are able to utilize the phosphorous stored in
this form. Enviropigs are able to convert phytate to inositol 2-monophosphate or inositol,
decreasing the need for phosphorous dietary supplements and lowering the phosphorous
concentration excreted into the environment.
15. Transgenic salmon have been produced with enhanced growth rates and better stress
and disease tolerance. Inserting an “all-salmon” growth hormone makes transgenic fish
that grow 3-5 times larger than wild type fish in a shorter period of time. Decreasing the
time it takes to produce full grown salmon will theoretically decrease production costs
and pollution, improving aquaculture.
CHAPTER 5
1. The sequence with accession number NM_000492.3 is the mRNA for Homo sapiens
cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family
C, member 7) (CFTR).
2. This protein is 100% similar to several huntington disease protein sequences in the
database.
10. In humans, the number of proteins outnumbers the number of genes because 60-70%
of human genes undergo some type of alternative splicing. This means that 30,000 genes
can produce approximately 85,000 proteins.
11. The principal feature of 2D PAGE is that proteins are separated first by one
characteristic and then by another (i.e. isoelectric focusing point and size). 2D PAGE has
high resolving power and is used to isolate individual proteins. Proteins are separated by
isoelectric focusing point by applying the proteins to a pH gradient gel and then
introducing an electrical current. When the protein has arrived at the position in the
gradient where the molecule has no net charge, then it will stop. If the second dimension
is size, the proteins are again separated on a gel with electricity. When proteins are
denatured to their primary structure, SDS ions (sodium dodecyl sulfate) surround the
proteins, masking the inherent charge of the protein and providing their own charge
(which is proportional to size since the number of SDS molecules that can associate with
a protein is determined by the size of the protein). The electrical current then causes
proteins to migrate through the gel at a rate the is proportional to the charge provided by
the SDS. The smaller proteins are subjected to less electrical force and less drag through
the gel while larger proteins are subjected to more electrical force and more drag. In a
polyacrylamide gel, though, the force of drag cancels out the difference in electrical force
so that smaller proteins migrate faster than the larger ones.
14. The protein is human serum albumin. MatrixScience.com also identifies the protein
as human serum albumin (Top Score: 113 for ALBU_HUMAN, Serum albumin
OS=Homo sapiens GN=ALB PE=1 SV=2).