Biomedical Engineering Reference
In-Depth Information
They left behind a gift for the unconquered Trojans - a huge wooden horse. The Trojans of
Troy willingly pulled the horse into their impregnable city to celebrate. During the night,
from inside the deceptively silent horse, Greek soldiers emerged, opened the gates for the
rest of their army to enter and destroyed the city from within. The idea of entering a cell
with the help of the cell and then attacking it from the inside is even more ancient than this
story. It is the process used by viruses, plasmids, bacteria and parasites. By using viruses
and plasmids as the Trojan horse, rendered harmless, we can modify genes or introduce new
ones to make the cell die or survive longer, secrete proteins or switch off genes, differentiate
or not differentiate.
In this chapter we discuss some of the strategies for gene delivery and give detailed
protocols on adeno-associated virus (AAV) as a gene delivery method which is safe, reliable
and long lasting.
12.1.1 General strategies for gene therapy: Basic methods
12.1.1.1 Transgenics
Studies using transgenics have revealed which genes are important or vital. Transgenic
mice with genes knocked out, or genes 'knocked in' to increase the number of copies of
genes [1], are the fodder of gene studies in living animals. They have been very useful
and practical for studying the role of specific genes producing specific human proteins. The
method involves harvesting embryonic stem cells from the inner cell mass of the blastocyst.
A desired gene is made, using recombinant DNA (rDNA), and inserted in a vector together
with promoter sequences to regulate the gene expression. A neo
r
gene, which is resistant
to lethal effects of neomycin, is added to the DNA cassette. A thymidine kinase gene
(tk) is also added. Those cells that fail to take the vector inside their walls can be killed
by neomycin. A few of the remaining cells allow the vector in, but the gene is inserted
randomly. Cells with random insertion of the gene are killed by gangcyclovir. The presence
of Tk phosphorylates gangcyclovir, which protects the gene. That leaves only those cells
in which homologous recombination has occurred. The normal gene has been knocked
out and a new, specified gene knocked in. These cells are then injected into a blastocyst
and implanted in the uterus to produce offspring that can be bred for more generations of
animals lacking a gene or possessing a new gene. If the new gene is nonfunctional (i.e.
a null allele), the function of the former gene may be revealed through breeding the mice
with the knockout gene to homozygosity. If the gene knocked out is absolutely essential
then it may be embryonically lethal.
Ideally, the function of the missing gene should be as obvious as if a limb had been cut
off. In actuality, several things can happen. The knocked out gene may prevent the embryo
from developing (it is embryonically lethal), or the missing gene is fully compensated by
other genes or subtle changes occur in development or in different organs also that the
effect is not obvious. Nevertheless, the technique has had a huge influence on revealing
functional effects of proteins, especially where antibodies have not been developed. The
opposite of knocking in copies of a gene has been used to reveal mechanisms of diseases
caused by overexpression of a protein [1]. The transgenic animal approach requires going
through embryonic development. This limits the technique when a knocked out gene is
embryonically lethal. However, a method first used by Gu
et al
. [2] is able to induce the
same mutation and avoid lethality.
