TROPLIST: Xeno Affy data ortholog conversions, combining insulators and SceI transgenesis, Trop Gal lines

[Kristen Kroll]

Hi all, 
I apologize in advance for the length of this message, but we have run into
a few issues where suggestions/information that anyone in the community has
would be very welcome:

1.  Large scale Xenopus gene-list correlation to gene symbols for mammalian
orthologs/nearest homolog.

Does anyone know of a program that can do a large scale correlation of
Xenopus laevis or tropicalis Unigene #s (or other Xenopus gene IDs, Affy
tags) to the official gene symbol of their mammalian ortholog or nearest
homolog?  

It would seem this should be SO simple to do, but...The annotation available
through Affymetrix for their Xenopus laevis arrays has improved, but it does
not facilitate defining gene symbols of mouse/human orthologs for Xenopus
genes on a large scale.  I have spoken with Affy tech support, and they say
that the consortia arrays (like the laevis arrays) do not yet have this
feature, and using their site/annotation queries engine we were not able to
find a way to look this up.

The many other "gene/ortholog conversion" tools I find on the web will not
accept Xenopus genelists, or (for programs like DAVID Bioinformatics), will
only convert one Xenopus gene ID to a different one, but not to the
ortholog.  It is frustrating, since this information exists and must be
built into sites like Metazome or XGI, but there does not seem to be a web
tool to batch search and bulk extract it, unless I have just missed finding
this.  

For single genes (or small lists) one can of course use the Unigene database
(NCBI), clicking on ortholog links for mouse/human for our Xenopus genes
(where these exist) and collecting the gene symbol for the ortholog.  This
"gene-by-gene" approach is impractical for large datasets however, and it
seems there must be a web-based tool that will this information from the
appropriate databases, and match it to the Xenopus Affytags/unigene#s, but I
have been unable to find one...if anyone has information or suggestions, I'd
greatly appreciate it...

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 transgenes 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.  Available frogs?  Good egg layers and/or Gal-GR transgenic line?

We are curious to see whether anyone has (a) Gal4-GR expressing tropicalis
transgenic driver line and/or (b) some wild-type (non-transgenic) animals
that are proven egg layers that they would be willing to send us.

We have been trying to use the NASCO purchased tropicalis for experiments
(injections to make transgenic lines) and so far it appears we often have
the bane of poor eggs. Our water hardness is relatively high and our
feeding/care regimen in our laevis facility results in females that lay
quality eggs consistently, so I suspect this is the "store bought animal"
problem with the tropicalis.  Along these same lines, does anyone know what
the current status of the proposed stock center (at the MBL?) is.

Many thanks for any information/suggestions,
Kris

______________________________________

 
Kristen L. Kroll, Ph.D
Associate Professor
Washington University School of Medicine
Dept. of Molecular Biology and Pharmacology
Campus Box 8103
660 S. Euclid Ave.
St. Louis, MO  63110

ph:  (314)-362-7045
FAX:  (314)-362-7058
email:  kkroll@wustl.edu

 

 


 

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