4th edition
CHAPTER 10
1. The IFN protein can be purified and used to used to make an antibody. The antibody
can then be used to screen a gene expression library, meaning that you clone and express
a cDNA library for the human cell line and then screen the entire library. The cloning
vector needs to have a promoter and you have to add ribosomes for in vitro translation,
and then each clone can be screened with the antibody. Alternatively, you could try to
sequence the purified protein and then use the protein sequence to create non-degenerate
oligonucleotide primers. You could then screen a cDNA library with the oligonucleotide
probe. The cDNA from the isolated clone could then be transferred to an expression
vector.
4. Two methods have been used to prolong the half-life of human growth hormone. The
first strategy involves the addition of a linker peptide with four repeats of [Gly4Ser]
between the extracellular domain and the normal protein. This construct dimerizes and
inactivates the molecule, allowing for a slow release of the active protein. The second
strategy fused the C-terminal end of human growth hormone to the N-terminal end of
human serum albumin. The fusion protein, Albutropin, has a longer half-life thanks to the
particular stability if the albumin protein.
5. Cystic fibrosis increases susceptibility to bacterial infections in the lungs. The lysed
cells release DNA and the live bacteria release alginate into the lungs, building up mucus
and constraining breathing. Dnase 1 and alginate lyase are useful in treating cystic
fibrosis because DNase1 hydrolyzes long DNA pieces into fragments, decreasing the
viscosity and adhesivity of the mucus. Alginate lyase can liquefy alginate which also
clears the lungs and improves breathing.
10. The Fv fragment contains all of the antigen-binding activity of the antibody, so
coupling an enzyme to an Fv fragment or to a monoclonal antibody targets the enzyme to
a particular antigen. These dual-function molecules can bind specifically to a target and
deliver a cellular toxin, some other enzyme activity, or a drug.
19. The XenoMouse produces only human antibodies and cannot produce any mouse
ones. This was accomplished by inactivating all of the mouse antibody production
machinery in one mouse line and by inserting the human antibody loci onto the mouse
chromosome in another line. The genes for the light and heavy chains were cloned into
YACs and then introduced into mouse embryonic stem cells. The two mouse lines were
then crossed and breed to select for the mice that only produce human antibodies.
24. Using antibodies against cancerous cells is difficult because most antigenic
determinants on cancerous cells are also present on non-cancerous cells. If a cancer
chemotherapy treatment induces the production of membrane bound proteins that are
unique to cancerous cells, then an antibody coupled to a cellular toxin can be used in
conjunction with this treatment to target and destroy only the cancerous cells.
CHAPTER 11
1. Psoriasis is a skin disease that is caused by uncontrolled epidermal growth. An
insulin-like growth factor is implicated in the disease. Antisense oligonucleotides can be
used to treat psoriasis. Antisense oligonucleotide therapy depends on the hybridization of
the oligonucleotide to the mRNA to form a double-stranded complex, preventing the
translation of the mRNA into an effective protein.
2. Ribozymes are naturally occurring catalytic RNA molecules that bind to a site on
RNA and then cleave it. Ribozymes are used as a therapeutic agent by genetically
engineering them to recognize a particular site on the target and then inactivate the
molecule by cutting it up. Ribozymes are often degraded before they reach their target,
but in theory, they are effective in disorders like cancer, inflammatory conditions, viral
and parasitic infections where overproduction of a protein is involved.
3. Interfering RNAs rely on the human and plant's natural response to the presence of
dsRNA in the cell. RNAi may have evolved to protect eukaryotes from viral infections
and accumulation of transposons. RNAi works as a therapy because dsRNA or siRNA
molecules complementary to the target RNA you want destroyed can be added to the
cells. The dsRNA are cleaved by Dicer into single-stranded short, small interfering RNA
nucleotides (siRNA). The antisense siRNA then binds to any complementary RNA
strands that it encounters. The RNA-induced silencing complex (RISC) includes the
antisense siRNA and cleaves the mRNA. The destroyed mRNA cannot be translated and
therefore the protein has been eliminated. RNAi has been directed against herpes simplex
virus type 2, hepatitis B and C viruses, Huntington disease, metastatic cancer,
transforming growth factor receptor 2 and other diseases.
6. Interfering RNA can be delivered to particular cells by using lipids, bacteria, collagen,
antibodies or aptamers. If the siRNA is attached to cholesterol and then injected into the
blood stream, the cholesterol-siRNA is taken up by various tissues (liver, hear, lungs,
etc.). Inside the tissue, the sense strand is destroyed, releasing the antisense siRNA which
is needed for RNAi. Nonpathogenic bacteria can also be transformed into a vector
carrying the gene for the shRNA molecule (under a bacterial promoter), and also the
genes to release genetic material from vesicles. This means that the bacteria can be
administered orally, produces many copies of the vector in the body of the patient, and
then releases the shRNA from the vesicles into the patient's body. The collagen-coupled
method uses positively- charged collagen fragments which are attracted to the negativelycharged siRNA. The collagen protects the siRNA from being degraded and the
atelocollagen-siRNA complexes can be injected locally. The antibody method relies on
the fusion of the antibody gene to the gene for protamine which binds negatively-charged
siRNA because of its positive charge. The antibody method directs the siRNA right to the
target site. Aptamers also bind specifically to a target antigen and can direct the siRNA to
the target. The entire aptamer-siRNA can be synthetically made.
8. The key attribute of therapeutic gene delivery is specificity of the targeting so that the
therapeutic gene makes it to the tissue that needs it. Systemic dosing often leads to
serious side effects. Although viral delivery systems are effective, there are several safety
concerns, and newer methods have been developed. The therapeutic agent needs to enter
the correct cell without being degraded, must meet with the target RNA, must specifically
bind only the target RNA and must reach enough cells in the target tissue to actually
affect the patient.
3rd Edition
Chapter 10
1. mRNA was isolated from the cells that can produce increased levels of the interferon.
It is then converted to DNA and inserted into plasmid vectors to create a cDNA library.
The vectors were transformed into E.coli and the clones divided into 12 pools. Based on
isolated interferon protein, a crude mRNA sequence could be determined. This would be
introduced into the 12 pools in order to hybridize with the desired DNA. The pools that
appeared to contain the correct mRNA sequence were divided further and rescreened
until a complete cDNA for IFN was found.
4. In cystic fibrosis, thick mucus accumulates in the respiratory track. One component of
this is DNA from bacterial calls and leukocytes that have broken open. Since DNase I
hydrolyzes long DNA chains into shorter pieces, it can reduce the viscosity of the mucus.
The other main component is alginate, a polysaccharide polymer excreted by the bacteria.
Alginate lyase breaks apart the polymer, also decreasing viscosity.
10. Humanize antibodies are antibodies where most of the molecule contains human
DNA sequences. The only mouse DNA present is in the complementarity-determining
regions (CDRs). If foreign antibodies from another species are used, there is a chance
that someone could be allergic to them. In addition, patients will develop their own
antibodies against the ones that have been given to them. Due to a lack of human
myeloma cells and even if there were, the antibodies produced by them would need to be
taken from human spleens, human monoclonal antibodies aren’t easily created. The
combination of the mouse and human antibody segments creates an antibody that is
highly specific, safe for humans, and can be engineered in mice.
17. Psoriasis is a disease characterized by uncontrolled epidermal growth that is possible
influenced by increased levels of insulin-like growth factor I receptors. Using an
antisense cDNA clone for the insulin-like growth factor I receptor, antisense RNA can be
created. When added to cells infected with psoriasis, it will bind to the cellular mRNA
that codes for the receptors and cause translation not to proceed. The decreased number
of receptors will lead to a reduced skin thickness of the lesions and decreased receptor
protein.
18. Ribozymes are RNA molecules that can bind and cleave other strands of RNA. The
binding and catalytic domains are separate, and so the binding domain can be engineered
to have any sequence that matches up with the target pathogen. As long as the sequences
match, the ribozyme will bind to the target DNA and cleave it at a specific spot.
19. Interfering RNAs are segments of double stranded RNA that are added to animal
cells, resulting in a loss of expression of the gene which the RNA is derived from.
Double stranded RNA would be created using cDNA for the gene that is being targeted.
When added to a cell, a specific nuclease binds to the dsRNA and causes it to be cleaved
into shorter pieces (which are single stranded). These single stranded pieces will
hybridize to the native mRNA coding for the same gene. Since the nuclease is still bound
to the RNA, the mRNA will be cleaved as well.
21. A therapeutic gene delivery system must be able to target a specific cell, protect the
DNA from degradation, direct DNA to cell nucleus, be expressed for a decent length of
time, and be noninfectious or too invasive.