A retained selection cassette increases reporter gene expression without affecting tissue distribution in SPI3 knockout/GFP knock‐in mice

Abstract

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.

A single-vector EYFP reporter gene assay for G protein-coupled receptors

“…It has been reported that retained high efficiency promoter, PGK (glycolytic enzyme phosphoglycerate kinase) could increase reporter gene expression when promoter and expression gene were located in a one transfection cassette [24]. Similarly, upon transfection, we observed a few cells (~0.1 %) presenting extremely strong constitutive EYFP signal in cells transfected with the single vector presented here.…”

Novel tumor necrosis factor‐knockout mice that lack Peyer's patches

“…3C). Opposing effects of neo cassettes on the expression of neighboring genes have previously been reported [45][46][47].…”

Aire-Deficient C57BL/6 Mice Mimicking the Common Human 13-Base Pair Deletion Mutation Present with Only a Mild Autoimmune Phenotype

“…Irrespective of such unresolved issues, our stable mutant protein, which contains the integral HSR, nuclear localization signal (NLS), SAND, and truncated PHD1 domains, closely emulates the common 13-bp deletion in human APECED patients for which several studies strongly suggest the expression of a stable truncated protein (27)(28)(29). Second, the targeting vector used in the deletion of exon 6 and premature termination of the Aire protein at the end of the SAND domain in Peltonen's mice has a selection cassette that was not removable, raising the possibility of affecting the expression of neighboring genes (51)(52)(53)(54)(55)(56)(57). Third, our model allows previously unappreciated insights into the role of Aire in the maintenance of normal fertility.…”