Biomedical Engineering Reference
In-Depth Information
12.1.1.2 Cre/Lox P System
To knockout a target gene in specific cell groups or tissue, in adult animals, the Cre/lox P
system is a suitable technique. It is based on the viral bacteria phage P1, which produces
Cre, a recombinase enzyme. Cre cuts its viral DNA into packages. Cre cuts all the DNA
out between two separate lox P sites. The DNA ends, which each have a half lox P site,
are then ligated by the recombinase. Gu
et al
. [2] used this principle with a strategy of a
conventional transgenic mouse, in which the Cre transgene plus a promoter was inserted by
homologous recombination in a cell-specific type. This mouse was crossed with a second
mouse strain that had a target gene flanked by two lox P sites. In the offspring the target
gene was only deleted in those specific cells that contained Cre. The target gene remained
functional in all the other cells and the animals survived development, so the function of
the targeted gene in specific cells could be studied.
More recent developments have made the technique less laborious to use [3, 4]. An
example is a study by Sinnayah
et al
. [4], who made transgenic mice with lox P insertions
flanking the gene for angiotensinogen. Angiotensinogen is a substrate for the enzyme renin
and is one of the critical components for the synthesis of the peptide angiotensin. Instead
of making a separate strain of Cre mice and proceeding with breeding, they simply injected
Cre into the floxed mice. This had the advantage of not only being time saving, but also of
opening up a new way to study genes with site-directed gene ablation in specific cells. As
they were working on the brain they were able to pinpoint anatomically a very small brain
structure. Their study addressed a long simmering debate of whether the brain makes its
own angiotensin [5, 6] or whether the angiotensin that has been found in the brain is taken
up from angiotensin in the blood. By injecting Cre into a brain structure they showed that
angiotensin synthesis could be blocked and, therefore, is made in the brain.
12.1.1.3 Antisense mRNA
To inhibit synthesis of proteins by inhibiting gene translation there are two methods: anti-
sense mRNA and RNA interference (RNAi). Antisense was discovered in 1977 [7], but it
was not until 1993 that its potential for inhibition by
in vivo
delivery was clearly demon-
strated. Antisense to neuropeptide Y Y-1 receptor was injected into the rat brain and the
injected animals showed a decrease of behavioral anxiety [8]. Antisense to angiotensinogen,
angiotensin-converting enzymes and angiotensin type 1 receptor genes was injected into the
brains of spontaneously hypertensive rats and they showed decreases in high blood pressure
[9]. Antisense is based on the fact that mRNA is in the 'sense' direction from 5' to 3'.
Antisense is a limited sequence of DNA in the antisense direction 3' to 5' designed from
knowing the sequence of a target gene. Antisense oligodeoxynucleotides (AS-ODN) are
usually built around the initiation codon of a gene (the AUG start site) but may be shorter
than the full-length gene. This is because the AS-ODN binds to part of the appropriate
mRNA sequences and prevents the mRNA from translating the protein it would otherwise
produce.
For gene modification within a cell with antisense a viral vector can be fitted with DNA
in the antisense direction. We have designed these in the adenoassociated virus and shown
them to have long-lasting inhibitory effects on designated cell protein synthesis [10].
Antisense inhibition, although widely used in research and approved for some clinical
treatments [11], is not perfect. When antisense is put into a cell it is competing with the
cells' own mRNA copying machinery. The presence of AS-ODN may actually increase the
