Transcription profiling by array of mouse wild type and Prss16 knockouts

Transcription profiling of Prss16 Tssp can be used to evidentiate further endopeptidase genes candidate to self-peptide generation in the thymus. All mice studied were C57BL/6 background and KO (knockout) Prss16-deficient mice were previously obtained by homologous recombination in embryonic stem (ES) cells of a targeting vector carrying Neo resistance gene marker, which has allowed replacement of exons 8 to 12 of the Prss16 gene in KO mice (data not shown). Briefly, one properly targeted ES clone was injected into BALB/c blastocysts to generate chimeric mice. Chimeric males were mated to C57BL/6 females to generate heterozygous pups in which the Neo selection cassette had been excised. Mice heterozygous for the mutation,originally on mixed 129/Sv x C57BL/6 genetic background, were intercrossed to generate homozygous mutants (Prss16-/-), WT (Prss16+/+) and heterozygousmutants (Prss16+/-) littermates. Genotype analysis was performed on genomic DNA from tail biopsies using PCR primers F (5' GCCTGACACAAGTCGCCATAGG 3'), R1 (5' CCAGTTCCTCCCTCAGCACAG 3') and R2 (5' CCAGTAAGAGTGAGGTCCAGAC 3'). The WT Prss16 allele was visualized as a 600 bp fragment using the F-R1 pair of primers, whereas the mutant allele was visualized as a 447 bp fragment using the F-R2 pair of primers. Absence of mRNA expression in the thymus of Prss16-/- mice was confirmed by northern-blot using a cDNA probe (data not shown). The Prss16 deficient mice were crossed onto a C57BL/6 background for eight generations and the thymi of the resulting mice were used for analysis.对Prss16 Tssp的转录谱分析（transcription profiling），可用于进一步鉴定可在胸腺中介导自身肽生成的内肽酶候选基因。本研究中所用小鼠均为C57BL/6背景。此前通过携带新霉素（Neo）抗性基因标记的靶向载体（targeting vector），在胚胎干（embryonic stem, ES）细胞中开展同源重组（homologous recombination），成功构建Prss16缺陷型敲除（knockout, KO）小鼠；该重组策略可使KO小鼠中Prss16基因的第8至12号外显子被替换（相关实验数据未展示）。简要而言，将1株正确靶向的ES细胞克隆注射至BALB/c小鼠囊胚（blastocysts）中，以制备嵌合小鼠（chimeric mice）。将嵌合雄性小鼠与C57BL/6雌性小鼠交配，获得已切除新霉素抗性筛选盒的杂合子幼鼠。最初饲养于129/Sv×C57BL/6混合遗传背景下的该突变杂合子小鼠，经互交繁殖得到纯合突变型（Prss16-/-）、野生型（wild type, WT，Prss16+/+）及杂合突变型（Prss16+/-）同窝小鼠。采用聚合酶链式反应（polymerase chain reaction, PCR）引物F（5' GCCTGACACAAGTCGCCATAGG 3'）、R1（5' CCAGTTCCTCCCTCAGCACAG 3'）及R2（5' CCAGTAAGAGTGAGGTCCAGAC 3'），对尾组织活检（tail biopsies）获取的基因组DNA进行基因型鉴定。使用F-R1引物对可扩增出600 碱基对（base pair, bp）的条带，对应野生型Prss16等位基因；使用F-R2引物对可扩增出447 碱基对（base pair, bp）的条带，对应突变型等位基因。通过Northern印迹（Northern blot）结合互补DNA（complementary DNA, cDNA）探针检测，证实Prss16-/-小鼠胸腺中无Prss16的mRNA表达（相关实验数据未展示）。将Prss16缺陷型小鼠与C57BL/6小鼠回交8代，随后取所得小鼠的胸腺用于后续实验分析。