Scarff Katrina L, Ung Kheng S, Sun Jiuru, Bird Phillip I
Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia.
Genesis. 2003 Jul;36(3):149-57. doi: 10.1002/gene.10210.
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.
人丝氨酸蛋白酶抑制剂，蛋白酶抑制剂6（PI-6/SERPINB6），是一种在许多组织中表达的蛋白酶抑制剂。它能抑制大量蛋白酶，包括粒细胞和单核细胞中的组织蛋白酶G。为了确定PI-6的时空分布，构建了小鼠模型，其中PI-6直系同源基因SPI3（Serpinb6）的外显子2被绿色荧光蛋白（GFP）报告基因取代。这使得GFP受SPI3基因的调控元件和起始密码子控制。新霉素选择盒两侧是loxP位点，以便从靶向等位基因中切除。杂合子和SPI3缺陷小鼠中的GFP表达准确反映了SPI3在所有测试器官中的组织分布，并允许对表达水平进行精确比较。有趣的是，靶向小鼠中保留新霉素盒导致白细胞中GFP增加2至10倍，但不影响组织特异性表达模式。这是选择盒保留特异性增加靶向小鼠中报告基因表达的首个例子，并强化了必须去除选择盒以避免对报告基因表达模式产生混淆影响的观点。
