Restrictase free generation of targeting vectors for disruption of complex mouse genes.
Molecular cloning of targeting vectors (TgVs) is a prerequisite procedure for gene disruption in embryonic stem cells. In cases where target genes display complex features (e.g., gene overlap, alternative exon usage), TgVs must mediate deletions with very high precision to prevent unwanted effects. This is often difficult to achieve by procedures using restriction endonucleases and DNA ligases. Therefore, to prepare TgVs for inactivation of two complex genes of immunological interest: PTPRF and NWC, we employed an alternative method, which involves engineering bacterial artificial chromosomes (BACs) by inducible, plasmid encoded "Red/ET recombinase" expression system. Here, we report rapid and efficient construction of PTPRF and NWC TgVs without using restriction endonucleases.
Miazek
Arkadiusz
A
Department of Tumor Immunology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R Weigla Wroclaw, Poland. arek@iitd.pan.wroc.pl
Cebula
Anna
A
Skwarek
Martyna
M
Cebrat
Małgorzata
M
Kisielow
Pawel
P
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics; D020868:Gene Silencing / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics*; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D011956:Receptors, Cell Surface / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D015280:DNA Restriction-Modification Enzymes; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D017027:Protein Tyrosine Phosphatases; C000709129:Ptprf protein, mouse; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2