Urs Sumithra, Harrington Anne, Liaw Lucy, Small Deena
Center for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, Maine 04074, USA.
Transgenic Res. 2006 Oct;15(5):647-53. doi: 10.1007/s11248-006-9000-z. Epub 2006 Sep 2.
Mouse strains expressing the site-specific Cre recombinase facilitate conditional ablation or activation of genomic sequences when one or several exons of a gene of interest are flanked by loxP sites. Recently, several strains targeting Cre expression to adipocytes have been developed using promoter sequences from the aP2 (Fatty Acid Binding Protein 4, FABP4) gene for adipose tissue-specific gene expression studies. aP2/FABP4 is predominantly expressed in adipose tissue, and while this promoter provides adipocyte-restricted expression postnatally, its expression throughout embryonic development had not been previously characterized. In this report, we demonstrate that the aP2-Cre transgene is expressed and consistently localized within the embryo from mid-gestation stage 9.5 dpc. By 15.5 dpc, beta-gal activity was detected primarily in the brown adipose tissue, trigeminal ganglia, dorsal root ganglia, cartilage primordia and vertebrae. Immunofluorescence staining for Cre recombinase and FABP4 protein showed a corresponding staining pattern similar to that of beta-gal, confirming that Cre recombinase was produced in the transgenic line at late stages of development, and overlapped with endogenous aP2/FABP4 production. Further, fat-specific oil red O staining of tissue sections validated the presence of lipids in the stained tissues indicating that adipocytes and/or adipocyte-like cells were indeed present in these tissues. This is the first report to our knowledge to describe and confirm aP2/FABP4 promoter expression in this transgenic line during development in the mouse embryo and indicates that aP2/FABP4 expression occurs not only in mature adipocytes, but has a wider embryonic expression pattern than previously appreciated.
当感兴趣基因的一个或几个外显子两侧为loxP位点时,表达位点特异性Cre重组酶的小鼠品系有助于基因组序列的条件性切除或激活。最近,利用aP2(脂肪酸结合蛋白4,FABP4)基因的启动子序列开发了几种将Cre表达靶向脂肪细胞的品系,用于脂肪组织特异性基因表达研究。aP2/FABP4主要在脂肪组织中表达,虽然该启动子在出生后提供脂肪细胞限制性表达,但其在整个胚胎发育过程中的表达此前尚未得到表征。在本报告中,我们证明aP2-Cre转基因在妊娠中期9.5 dpc的胚胎中表达并始终定位于胚胎内。到15.5 dpc时,主要在棕色脂肪组织、三叉神经节、背根神经节、软骨原基和椎骨中检测到β-半乳糖苷酶活性。对Cre重组酶和FABP4蛋白进行免疫荧光染色,显示出与β-半乳糖苷酶相似的相应染色模式,证实Cre重组酶在发育后期的转基因品系中产生,并与内源性aP2/FABP4的产生重叠。此外,组织切片的脂肪特异性油红O染色证实了染色组织中存在脂质,表明这些组织中确实存在脂肪细胞和/或脂肪样细胞。据我们所知,这是第一份描述并证实aP2/FABP4启动子在小鼠胚胎发育过程中在该转基因品系中表达的报告,表明aP2/FABP4不仅在成熟脂肪细胞中表达,而且其胚胎表达模式比以前认识的更广泛。