dictyNews
Electronic Edition
Volume 37, number 12
November 11, 2011

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Abstracts
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Highly effective removal of floxed Blasticidin S resistance cassettes 
from Dictyostelium discoideum mutants by extrachromosomal 
expression of Cre

Joern Linkner, Benjamin Nordholz, Alexander Junemann, Moritz Winterhoff 
and Jan Faix

Institute for Biophysical Chemistry, Hannover Medical School, Hannover, 
Germany


EJCB, in press

The inactivation of proteins in cells is inevitable to study their physiological 
role in various cellular processes. In contrast to strategies to alter the 
amount of active proteins in cells, only a gene knockout guarantees 
complete removal of the protein of interest. For Dictyostelium discoideum 
cells, the gene replacement construct typically consists of a Blasticidin S 
resistance (Bsr) cassette flanked by fragments of the target gene to allow 
insertion by homologous recombination. More advanced knockout constructs 
additionally carry loxP sites on both sides of the Bsr cassettes for subsequent 
removal of the selection marker by transient expression of Cre recombinase, 
thus allowing generation of multiple knockouts using just a single selection 
marker. However, due to its design, the available neomycin selection-based 
Cre expression plasmid occasionally tends to integrate into the genome and 
also yield only a moderate number of transfectants in liquid media. In some 
cases, for instance in SCAR-null cells, it was not possible to remove the 
Bsr cassette without stable integration of the Cre expression vector into the 
genome. To circumvent these difficulties we designed the extrachromosomal 
Cre-recombinase expression vector pTX-NLS-Cre. We verified the greatly 
improved efficacy of this novel Cre-loxP approach by removal of the Bsr 
cassette in five different cell lines including the SCAR-null mutant. By 
consequence, this vector will be a highly valuable means for the rapid 
generation of single or multiple mutants remaining sensitive to the most 
reliable selection markers Blasticidin S and neomycin.


Submitted by Jan Faix [[log in to unmask]]
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Characterization of NE81, the first lamin-like nucleoskeleton protein 
in a unicellular organism

Anne Krueger, Petros Batsios, Otto Baumann, Eva Luckert, Heinz Schwarz, 
Reimer Stick, Irene Meyer*, Ralph Graef*

Dept. of Cell Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 
14469 Potsdam-Golm, Germany

	
Mol. Biol., Cell in press

Lamins build the nuclear lamina and are required for chromatin organization, 
gene expression, cell cycle progression and mechanical stabilization. Despite
these universal functions, lamins have so far been found only in metazoans. 
We have identified a protein NE81 in Dictyostelium, whose properties justify 
denomination as a lamin-like protein in a lower eukaryote. This is based on its 
primary structure, subcellular localization, regulation during mitosis, and the 
requirement of the C-terminal CaaX box as a post-translational processing 
signal for proper localization of the protein. Our knock-out and overexpression 
mutants revealed an important role for NE81 in nuclear integrity, chromatin 
organization and mechanical stability of cells. All our results are in agreement 
with a role of NE81 in formation of a nuclear lamina. This function is 
corroborated by localization of Dictyostelium NE81 at the nuclear envelope in 
human cells. The discovery of a lamin-like protein in a unicellular organism is 
not only intriguing in the light of evolution, it may also provide a simple 
experimental platform for studies of the molecular basis of laminopathies.


Submitted by Ralph Gräf [[log in to unmask]]
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[End dictyNews, volume 37, number 12]