TROPLIST: TROPLIST summary of: combining insulators and SceI transgenesis, Trop Gal lines

[Kristen Kroll]

Here is a summary of some replies to these queries. Thanks again for the
information. (this is the second of two posts summarizing info/replies from
this thread)

2.  Does addition of insulators greatly improve expression levels of
transgenes in SceI meganuclease transgenesis?

We have been wondering whether or not we should try flanking
promoter-reporter cassettes with insulators (for example, beta-globin
After Dan Week's integrase paper).  Has this been tried by some labs in
combination with SceI meganuclease transgenesis and does it enhance
expression levels appreciably?

In our prior comparisons, transgenic embryos made by REMI transgenesis
expressed a given transgene at higher levels than SceI meganuclease
transgenics made with the same enhancer-reporter. This is probably because
the transgene inserts as multimers in REMI and one can boost the total
copy number higher by modulating the reaction conditions also.  Since we
Want to raise animals to make lines, the meganuclease method appears
favorable to get a higher rate of normal advanced development. Therefore,
anything that  would enhance the transgene expression level after SceI
transgenesis would  be very helpful.

3.  Gal-GR transgenic line?

>>  Hi Kris-
>>      The Gal4-progesterone receptor GLVP aka GalPR (Gal4 DNA-binding,
>>  VP16 activation, mutant RU486-responsive progesterone receptor
>>  ligand-binding domain) seem to work pretty well.  If Rob doesn't have
>>  them anymore we might be able to send you some ntubulinGalPr ones.
>>      Re bad eggs- my feeling is that hard water tends to be good; I
>>  think diet is key.  Whatcha feeding them?  We had excellent eggs with
>>  Tetra Reptomin (aquatic pet turtle food!) but then Tetra radically
>>  changed the formulation without so much as a by-your-leave.
>>
>>      I think you'd have been better off writing 4 separate queries...
>>
>>  con mucho gusto
>>  Lyle
>>

>Hi Lyle,
>Wow--thanks for the quick reply.  Maybe I was just being impatient on the
>bad eggs, we'd been through a number of bad egg days recently using newish
>NASCO animals (they've been sitting in the facility for a while too); so I
>was worrying we'd never see good ones. But today all looks much better...go
>figure (maybe the effects of moving to a new tank).
>
>I saw your note about the Reptomin being good (before they changed it)
>either on your website or in the archives and was going to see if I could
>find that here.  Maybe we can do a direct comparison. Right now we feed
>NASCO chow--Our laevis gals have generally given nice eggs for years on
>that.  Maybe trops need something richer/different? Had you cross-compared
>those feeds? We had a few issues with the new trop facility, in its early
>days (have had it up for about 1 yr now); usually alarms have gone off and
>they catch any water quality issue before it doses into the system, but I'd
>begun to wonder if we'd compromised these animals at some point when we had
>this spate of poor eggs.
>
>Thanks for the info about the GalPR line.  Depending on how we do on all
>this, I may bug you or Rob for it at some point. I'd been considering
>building a ubiquitous CMV-GalGR line, since I've used this successfully in
>transients w UAS plasmids. Not sure if there are silencing issues with
>ubiquitous promoters late, or if EF1alpha or something else may be better
>from this perspective. I seem to remember some discussion of silencing
>issues  in transgenic lines at the last frog meeting...will have to look
>into it.
>
>Our GalGR includes truncated DNA binding and activation domains; we also
>have a version that sounds like what you say--Gal DBD-VP16 activation
>domain-GR ligand binding domain; this showed some toxicity when injected as
>an mRNA, maybe because the VP16 AD is so active.   Have you ever tried a
>ubiquitous version of your GalPR; if so, would be curious which regulatory
>sequences you used?
>
>You are probably right, I should have split the queries, I seem to be on a
>lot of different "fact finding missions" at the moment...Thanks for helping
>with this one.
>
>Kris

Hi Kris-
    We just finished a diet trial in which Nasco chow did quite poorly
for both growth and egg quality (although the sample size for egg
quality was only two females for the five different feeds).
    The other thing I'd ask is whether the temperature has gotten
above 27C.  The frogs like it, but their eggs suck for months
afterwards.  We aim for 24-26C year-round.
   We haven't made or tested ubiquitous GalPr.  Rob might have.
Lyle

Hi Lyle--thanks for that info.  I will see if we can get some
tanks going on  the reptomin if that is your favorite from the trial (even w
the new formulation?), can't hurt.  We were going to use the NASCO sera
micron for tadpole culture (have been using some old NASCO tadpole brittle
we had), but if there is something you've found better let me know.

I will have to check whether we ever had an issue with temps down there. I
remember the same thing back in Berkeley in the late 80s w the  laevis;
therewas a heat wave and all the frogs in the greenhouse survived but then
at least in some entire tanks the females broke down and resorbed all their
oocytes (I was doing a bunch of surgeries to isolate oocytes at the time, so
it was pretty striking to get frog after frog w mushy/deteriorating
oocytes).  So we had no decent eggs for months and then
great ones when the fresh batches post heat-shock matured.

On our trops, I doubt it was hot, because of where the facility is in the
bldg., but we did have a few weird things go on this year as we tried get
 the Marine Biotech systems online and to establish homeostasis, I
think more water quality issues, alarms going off a lot.  Initially our UV
bulbs were not giving the dose they should have and we had rotifers living
in the system, frogs didn't mind but the animal guys did; Marine Biotech
fixed some of our glitches in a site visit and seems okay now (probably
shouldn't even be saying that...)

We had someone here build a facility database for logging all of the daily
information on water quality, daily/weekly/monthly maintenance for the
 marine biotech system, whenever the alarms trigger, feeding regimen, etc.
This was because I was tired of reverting to trying to remember after the
fact exactly what was going on if egg quality took a turn south.  Has been
 quite useful, the animal techs like having one place to record
what is being done down there (instead of the former paper logs) and so we
have pretty good records when something happens what it was and how that
affects the animals at different points later.

I should probably link it on our web site, so that anyone using the marine
biotech recirc systems could try a copy and we could make improvements.
Could still use some tweaking, but forhow expensive and complicated these
systems are, I was somewhat disappointed with what they were able to give us
as standard operating procedures for  care/maintenance/troubleshooting of
the various parts of the system.  I had  wanted to make it trivial to train
new animal care people into it, have anything on paper as easy checklists
and their information and they didn't have their SOPs organized in such a
way.
>>>
>>>  I'll have to check w Nick Hirsch also on the ubiquitous promoters; I think
>>>  he has looked into which are silenced in later development, which I'd
>>>  ignored in my 'early and transient assays' but will matter for lines.
>>>
>>>  If you have any other feeding suggestions let me know, and thanks for the
>>>  info. That is very helpful--Kris
>>>

Hi Kris
Try combining Reptomin with fish flake; that gave us the best results
and it's fairly easy to use.  Sera Micron is still our favourite for
tads (much better than tadpole brittle) esp if you grow tads in
bright light (the sera micron blooms and it's easy to keep the water
fresh with very low flow).  As they get bigger, they'll do well with
additional ground fishflake, and then flake + crushed reptomin for
tads.-Lyle

   re food, just bear in mind it may take 3mo+ for diet to improve egg
quality.  Reptomin formulation changed about 18 months ago, protein
content dropped by about 50%.


> Hi Kris,
> We've tried numerous enhancers with the insulators from Dan's lab and
> haven't found any increase in the level of expression, but rather a
> MODEST increase in the fidelity of expression. I think you are exactly
> right about REMI having higher expression levels due to the increased
> copy number. That being said, we do see a few tandem (or inverted)
> insertions in most cases with Sce which makes it somewhat difficult to
> figure out the insertion site using RACE or related methods. The
> biggest problem is the inclusion of the plasmid backbone which I am
> certain has its own effects on expression. We have considered trying
> to digest the transgene with Sce and cleaning it away from the
> backbone, but this didn't seem to have much effect in the early Sce
> papers in fish. Perhaps Tol2 is the way to go? Any other thoughts you
> might have on this topic would be welcome.
> 
> We have thought about making a hormone inducible Gal4, but haven't
> gotten around to it yet. I'd love to hear if you find anyone out there
> that has one.
> 
> Sorry I can't help you with the translation of gene names.
> 
> Kurt

Hi Kurt,
That information is all really useful.  Think I will not bother trying to
add the insulators then, as we try to cook up the perfect multicassette
vector to make lines. I'm not too concerned about insertion site, other than
checking enough transgenic individuals to see a consistent pattern and
discard individual founders that may have integration position effects. So
from what you say, we can probably continue doing our transgenesis exactly
as we have been and it will not make much difference.  I do w

By the way, regarding your prior post on ChIP in Xenopus (I don't know if
you got an answer on this), we have been doing a lot of ChIP on mouse brain
tissue and ES cells but not Xenopus embryos.  However, at the last Xeno
meeting I talked with Gert Jan Veenstra
http://www.ncmls.nl/gertjanveenstra/contact.htm; they are in the
netherlands. He said he was getting good chip-on-chip data from embryos and
offered to send protocols.  If you do get some useful info from him, could
you let me know/send it along to me also, if he's willing to share some
tips?  Right now the dearth of ChIP-quality/verified antibodies is still a
major hurdle to this for us, relative to doing the same expts in mammalian
systems.  Certainly would be nice if there were a resource for generating
such antibodies.  I know Gert had an active interest in this problem as well
when I spoke with him.  I don't know if some larger entity could be
convinced to generate these antibodies/or test existing for their
suitability in Xenopus (like someone at Nimblegen?) but certainly would be
wonderful if that could be arranged.

Best regards,
Kris

 

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