A Dicty rosa26 or the like site would be great. However, at least in 
mice and other mammalian systems including sheep, transgenic founders 
that are propagated based upon successful expression of the KI transgene 
show that the position of integration within the chromosome looks like a 
"salad". Hence the difference in phenotypes observed between mice 
transgenic for the same construct....at the site of integration...we see 
multiple insertions in either direction of mostly truncated transgenes 
but one transgene intact that allows for detection of founders. 
Partitioning of plasmid between dividing Dicty cells must be unequal and 
why there is significant "expression differences" from cell to cell from 
the same clone even when cell cycles are synchronized.

Although a sidetrack, CRISPR is definitely being developed for Dicty.

As Wolfgang mentions, epistatic effects and positional effect 
variegation in Dicty is still new.

On 2013-10-09 15:50, Chubb, Jonathan wrote:
> Wolfgang is correct, but we have recently been able to express 
> transgenes
> (usually just GFP) by knock-in into a handful of strongly expressed 
> genes.
>  It is not as bright as over-expression can be, but depending on the
> target site, we get considerably less variability in expression 
> between
> cells within a clone.  The idea is to find some kind of Dicty rosa26 
> or
> HPRT site.
>
>
>
> On 09/10/2013 05:06, "Uni.-Prof. Dr. Wolfgang Nellen"
> <[log in to unmask]> wrote:
>
>>To my knowledge there is no targeted gene insertion except
>>for disruptions and maybe mutagenesis of a gene.
>>There is still the problem that e.g. insertion points of
>>transgenes are rather difficult to determine (at least in
>>our hands).
>>This is documented by different phenotypes that you may
>>get with different independent clones. There is a lot of
>>epigenetics going on and we have only scratched the
>>surface.
>>Even with extrachromosomal vectors, phenotypes may vary.
>>No idea why!
>>Correct me if I am wrong!
>>wolfgang
>>
>>
>>On Tue, 8 Oct 2013 22:44:35 -0400
>>  "John B. Biggins" <[log in to unmask]> wrote:
>>> Thanks.  I'm a chemist & I'm writing up some stuff &
>>>need background.
>>>
>>> John
>>>
>>>
>>> On Oct 8, 2013, at 9:47 PM, Christopher West wrote:
>>>
>>>> Yes, it is done all the time. You might start with:
>>>>
>>>> Nat Protoc. 2007;2(6):1317-24.
>>>> Transformation of Dictyostelium discoideum with plasmid
>>>>DNA.
>>>> Gaudet P, Pilcher KE, Fey P, Chisholm RL.
>>>> Source
>>>> dictyBase, Center for Genetic Medicine, Northwestern
>>>>University, 676 North Saint Clair Street Suite 1260,
>>>>Chicago, Illinois 60611, USA.
>>>> Abstract
>>>> DNA-mediated transformation is one of the most widely
>>>>used techniques to study gene function. The eukaryote
>>>>Dictyostelium discoideum is amenable to numerous genetic
>>>>manipulations that require insertion of foreign DNA into
>>>>cells. Here we describe two commonly used methods to
>>>>transform Dictyostelium cells: calcium phosphate
>>>>precipitation, resulting in high copy number
>>>>transformants; and electroporation, an effective
>>>>technique for producing single integration events into
>>>>genomic DNA. Single integrations are required for gene
>>>>disruption by homologous recombination. We also discuss
>>>>how different selection markers affect vector copy number
>>>>in transformants and explain why blasticidin has become
>>>>the preferred selectable marker for making gene
>>>>knockouts. Both procedures can be accomplished in less
>>>>than 2 h of hands-on time; however, the calcium phosphate
>>>>precipitation method contains several incubations,
>>>>including one of at least 4 h, so the total time required
>>>>for the transformation is approximately 8 h.
>>>> PMID: 17545968 [PubMed - indexed for MEDLINE]
>>>>
>>>> -Chris West
>>>>
>>>>
>>>> On Oct 8, 2013, at 7:31 PM, "John B. Biggins"
>>>><[log in to unmask]> wrote:
>>>>
>>>>> Has anyone had any success in targeted insertion of
>>>>>genes within the Dicty genome?
>>>>>
>>>>> That is, you can stably insert the DNA you want in the
>>>>>region you choose?
>>>>>
>>>>> Thanks
>>>>>
>>>>> John
>>>>
>>>
>>
>>Wolfgang Nellen
>>Abt. Genetik
>>Univ. Kassel
>>Heinrich-Plett-Str. 40
>>34132 Kassel
>>Germany
>>

-- 
-------------------------------------------------
Michael A. Myre, PhD
Instructor of Neurology, Asst. in Genetics
Center for Human Genetic Research
Massachusetts General Hospital
Harvard Medical School
185 Cambridge St. Rm. 5.612A
Boston, MA 02114
Ph. 617-643-5536
[log in to unmask]
Website: http://chgr.org/index-faculty_myre.html
Website: 
http://www.massgeneral.org/neurology/researcher_profiles/myre_michael.aspx
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