Although a lot of the field is moving to working on TALEN's zinc fingers haven't yet been completely replaced.
Although zinc finger proteins do have context-dependent effects like you mentioned (you can't string together any two zinc fingers into an array) there are currently two different assembly methods for zinc finger arrays which work around this (OPEN and CoDA) by building arrays using zinc fingers that are known to work well in combination together or optimizing the zinc finger array at every step of construction. These processes are pretty long and cumbersome to just stringing together TALE's (TALENs are the the TALE protein + a nuclease domain) though which don't have any known context dependent effects.
BUT the size of the TALE protein arrays is a concern for a lot of researchers because for a zinc finger array, each individual zinc finger binds 3 base pairs of DNA but for a TALE array, each TALE only binds one base pair. As a result, a zinc finger array only needs 6 zinc finger proteins to bind to an 18 base pair sequence while a TALE array would need18 proteins to bind to an 18 bp sequence.
The field is definitely moving toward TALE's but zinc fingers will still be around for at least a little longer until more research on TALE's is done/the kinks get ironed out.
On Wednesday, September 26, 2012 11:37:05 AM UTC-4, Cathal wrote:
I studied Zinc Finger Nucleases long enough to know that they are a dead--
end. Seriously, six-feet-under dead end. They're already being replaced
by TALENs because TALENs aren't as Patent-encumbered.
Essentially, about half of the known zinc finger families are patented
to the hilt, and the patent troll that owns them charges in the 10,000s
for licenses. The remaining zinc fingers aren't sufficient for reliable
assembly; it turns out that, despite their glowing reputation for
"modular DNA targeting proteins", they are actually very hard to get
working correctly. You can add a zinc finger for "TAG" to a zinc finger
for "GAC" and have it totally fail to bind to TAGGAC, and instead bind
to TTAGWGAC, or something like that.
I haven't studied TALENs enough to know for sure yet, but I gather they
are somewhat more predictable. Being less patented is ALWAYS a boon, and
it seems that it's paying off.
As to how they work; when using them in-vitro, you can use them as
proteins are normally used; protein + buffer + DNA etc.
When used for gene therapy, it's a totally different situation; you
don't deliver the protein, you deliver a DNA agent that encodes the
protein. So, you design a plasmid containing your ZFN/TALEN, plus the
DNA you want to replace the chromosomal target with (it must have
significant homology to the chromosomal target to encourage crossover
after enzyme-cleavage), and you deliver that through electroporation, or
chemical treatment, or viruses.
The TALEN/ZFN gets transcribed/translated and, if you've got the
appropriate nuclear targeting peptides attached, gets sent back into the
nucleus, where it cuts the target site. Then, homology-directed DNA
repair (homologous recombination) leads to the target site getting
replaced with your alternative sequence in the plasmid at some efficiency.
If your replacement is designed not to contain the target site, this is
one-way and reasonably high efficiency, provided that the DNA-cleaving
TALEN/ZFN works as intended and gets into the nucleus. You have to
provide lots of plasmid to ensure there's plenty of template lying
around when the break occurs in the target strand.
Efficiency per targeted cell is good, but don't expect to transform
whole organisms, it will not happen. Efficiencies of transduction in
animals is very low for naked DNA, often less than 10%. When using
viruses, you can get higher efficiencies, but at the cost of high
specificity (only certain cell types get transformed) and potential
immune overreactions. Methods for non-naked-DNA, non-viral DNA delivery
include electroporation, high pressure delivery, and liposomal delivery.
Options for Viral delivery which aren't insanely risky to attempt on
humans include: Adeno-Associated-Virus (NOT Adenovirus). And that's it.
The others are highly risky in terms of immune response, and if you use
them as they are most often used, they also carry significant cancer
risks due to random-ish, gene-preference integration. AAV is remarkable
as it has such low immune stimulation effects, and it has a target site
in the human genome that seems to have very low risks of any cancerous
side-effects (provided it's unoccupied by wild AAVs).. but it has really
low capacity for DNA, so it's hard to deliver anything useful.
Naked DNA has low efficiency of transformation, but at least it's A)
Easy and B) More controllable; integration is only likely where you
direct it to occur, and immune reaction to naked DNA is generally mild,
particularly if you produce it via PCR or DAM/DCM negative strains of
E.coli, so it's not methylated in ways that the immune system treats as
suspicious.
On 26/09/12 08:54, Mega wrote:
> Hi,
>
>
> I read about zink finger nucleases, and that they may be used in living
> organisms to alter their genome (after birth, as an adult) .
>
> I was wondering how that was posssible, how do you get the enzyme in every
> cell of the body? Is it just injected in the blood stream and then gets
> distributed automatically?
> Or do you inject it into the organ needed? But how can it penetrate the
> cell wall? Is it so small that it fits through the celll membranes?
>
>
>
--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To post to this group, send email to diybio@googlegroups.com.
To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/_VhKl5zyM7AJ.
For more options, visit https://groups.google.com/groups/opt_out.
0 comments:
Post a Comment