A retained selection cassette increases reporter gene expression without affecting tissue distribution in SPI3 knockout/GFP knock-in mice.
The human serpin, proteinase inhibitor 6 (PI-6/SERPINB6), is a protease inhibitor expressed in many tissues. It inhibits a large number of proteases, including cathepsin G in granulocytes and monocytes. To determine the temporal and spatial distribution of PI-6, mice were generated in which exon 2 of the PI-6 ortholog SPI3 (Serpinb6) was replaced with a green fluorescent protein (GFP) reporter gene. This placed GFP under the control of the regulatory elements and initiation codon of the SPI3 gene. The neomycin selection cassette was flanked by loxP sites to allow excision from the targeted allele. GFP expression in heterozygous and SPI3-deficient mice accurately reflected the tissue distribution of SPI3 in all organs tested and allowed precise comparisons of expression levels. Interestingly, retention of the neomycin cassette in targeted mice resulted in 2-10-fold increases of GFP in leukocytes, but without affecting tissue-specific expression patterns. This is the first example of selection cassette retention specifically increasing reporter gene expression in targeted mice and reinforces the view that selection cassettes must be removed to avoid confounding effects on reporter gene expression patterns.
Scarff
Katrina L
KL
Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia.
Ung
Kheng S
KS
Sun
Jiuru
J
Bird
Phillip I
PI
D000818:Animals; D017931:DNA Primers; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins; D051379:Mice; D018345:Mice, Knockout; D009355:Neomycin / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics*
D017931:DNA Primers; D008164:Luminescent Proteins; D015843:Serpins; D049452:Green Fluorescent Proteins; D009355:Neomycin