Identification of genes characteristic of Zfp423-expressing perivascular cells in mouse epididymal adipose tissue

Committed perivascular preadipocytes were genetically labelled in transgenic mice expressing GFP under the control of the locus (BAC) for Zfp423, a gene controlling preadipocyte determination. The overall goal was to identify genes differentially expressed between perivascular (PDGFRbeta+) GFP+ cells and perivascualar (PDGFRbeta+) GFP- cells from mouse epididymal adipose tissue. Adipose stromal vascular fraction from epididymal fat of 5 week-old male Zfp423-GFP mice was labeled with fluorophore-conjugated antibodies recognizing PDGFRbeta, CD31, and CD45. Pdgfrbeta+; CD45/CD31- (herein Lin-) cells were then separated on the basis of GFP expression. GFP+ and GFP- from the PDGFRbeta+ Lin- perivascular population were collected by FACS into separate tubes. Triplicate biological samples were collected for RNA extraction and Illumina analysis.

在以调控前体脂肪细胞定向分化的基因Zfp423的细菌人工染色体（Bacterial Artificial Chromosome，BAC）位点为驱动元件表达绿色荧光蛋白（Green Fluorescent Protein，GFP）的转基因小鼠中，对定型血管周前体脂肪细胞进行了遗传标记。本研究的总体目标是从小鼠附睾脂肪组织中，筛选出血管周（血小板衍生生长因子受体β阳性，PDGFRβ+）GFP阳性细胞与同群PDGFRβ+GFP阴性细胞之间的差异表达基因。从5周龄雄性Zfp423-GFP小鼠的附睾脂肪中分离得到脂肪基质血管组分（Stromal Vascular Fraction，SVF），并使用识别PDGFRβ、CD31及CD45的荧光素偶联抗体进行标记。随后基于GFP表达水平，分离得到PDGFRβ+、CD45与CD31均阴性（本文中记作谱系阴性，Lin-）的细胞。通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS），将PDGFRβ+ Lin-血管周细胞群中的GFP阳性与GFP阴性细胞分别收集至独立离心管中。收集三份生物学重复样本用于RNA提取及Illumina测序分析。