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
>