A targeting vector for the Rosa26 gene was constructed using the recombineering approach and utilizing the Cre-lox system for future RMCE manipulations.  The long and short arm Rosa26 fragments (LA & SA) are 8.232 kb and 3.770 kb respectively and were derived from a RP22 BAC clone (#58-D17).  A 5.165 kb region between these arms has been replaced by the floxed tk-neo cassette, a puromycin-Δ-thymidine kinase fusion gene driven by the mouse phosphoglycerol kinase promoter (pUDTK) and a kanomycin resistant gene driven by the bacterial EM7 promoter (EM7-kan) flanked by minimal (34 bp) tandemly oriented lox71 and lox2272 sites (Cre-recombinase recognition sequences).  The construct also contains a diphtheria toxin negative selection cassette (pgk-DT-A).  It should be noted that the LA contains a single base difference in what is contained in this targeting vector plasmid and the predicted sequence derived from MGI database:  database predicts a 17 bp run of “A’s” at position 9274 of the targeting vector plasmid; the sequence data confirms only 16-A’s here in the plasmid.

 


Category
Plasmid
Record History
Added on February 10, 2009 at 11:06 AM by Gangula, Rama
Modified on September 5, 2013 at 1:00 PM by Magnuson, Mark
Shared with (contributions)
Public: Shared Collection

Vector Annotations

Vector Map NYNa6xd7.png
Genbank File pRosa26.LCA.gb
Backbone Vector pBluescript KS+
Construct Size 19,786 bp (approximate)
Bacterial Stock No
Storage Temperature -20°C
Stock Concentration 0.1 µg/µL
Addgene Not Provided

Source

Laboratory Mark Magnuson
Made byXuan Li
Stock Date February 10, 2009

Inventory Location

Private

Publications / Citations

  1. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice. Chen SX, Osipovich AB, Ustione A, Potter LA, Hipkens S, Gangula R, Yuan W, Piston DW, Magnuson MA (2011) Dis Model Mech 4(4): 537-47
    › Primary publication · 21324933 (PubMed) · PMC3124063 (PubMed Central) · Added on 11/8/2013

MeSH Terms

Alleles Animals Chromosomes, Artificial, Bacterial Embryo, Mammalian Embryonic Stem Cells Fluorescence Genetic Loci Genetic Vectors Luminescent Proteins Mice Mutagenesis, Insertional Organ Specificity Proteins Rabbits Recombinases RNA, Untranslated