PARP1 knockout mice

Poly(ADP-ribose) polymerase 1 (PARP1) is a highly conserved nuclear protein in multicellular organisms that modulates chromatin opening, and by doing so, it plays a significant role in gene expression regulation during development and differentiation. Despite its significance, all reported Parp1 total knockout mice strains are viable and fertile with no developmental anomalies. It was previously believed that functional redundancy with other PARP family members explain such a controversy. PARP2 is the main candidate to fulfill PARP1 functions in Parp1 knockout mice. Indeed, Parp1 Parp2 double knockout mice are arrested early in development and unable to proceed through gastrulation. However, while PARP2 has similar catalytic domain to PARP1, it lacks other domains that play important regulatory roles, thus making the lack of developmental problems in Parp1 mice total knockouts highly unlikely. To address this question, we checked the best investigated and publicly available Parp1 knockout mice strain. We found that Parp1 is still expressed in these mice, based on the presence of its mRNA transcript. Contrary to prior assumptions, we identified persistent mRNA expression in knockout mice, albeit at reduced levels. Detailed transcript analysis revealed an alternatively spliced PARP1 variant lacking exon two with introduced artificial cassette. Subsequent protein analysis confirmed the existence of a truncated PARP1 protein in knockout mice. We established embryonic stem cell lines from knockout mice and revealed the presence of pADPr. The decreased level of pADPr was detected in knockout ES cells with Western Blotting analysis, but immunofluorescence staining didn't detect any difference in distribution or level of pADPr in nuclei of knockout ES cells compared to control ones. Moreover, in double Parp1 Parg mutant ES cells the accumulation of pADPr greatly exceeded its amount in normal and even in hypomorph Parg mutant ES cells, suggesting the presence of functionally active PARP1. Therefore, our findings challenge the conventional understanding of PARP1 depletion effects. To check the presence of Parp1 on mRNA level in knockout mice we performed RNAseq analysis in testis of control and knockout mice.聚ADP核糖聚合酶1（Poly(ADP-ribose) polymerase 1，PARP1）是多细胞生物中高度保守的核蛋白，可通过调控染色质开放状态，在发育与分化过程中对基因表达发挥关键调控作用。尽管PARP1功能至关重要，但目前已报道的所有Parp1全基因敲除小鼠品系均存活且可育，无发育异常表型。此前学界认为，这一矛盾现象可通过PARP家族其他成员的功能冗余来解释，其中PARP2被认为是在Parp1敲除小鼠中替代PARP1功能的主要候选蛋白。后续研究证实，Parp1与Parp2双基因敲除小鼠会在发育早期停滞，无法完成原肠胚形成。然而，尽管PARP2与PARP1拥有相似的催化结构域（catalytic domain），却缺失了其他发挥重要调控功能的结构域，因此Parp1单基因敲除小鼠未出现发育问题的结论便难以成立。为解答这一问题，我们对研究最为深入且公开可用的Parp1敲除小鼠品系进行了验证。基于其mRNA转录本的存在，我们发现该品系小鼠中仍可检测到Parp1的表达。与此前的假设相反，我们在敲除小鼠中检测到了持续的mRNA表达，尽管其水平有所降低。详细的转录本分析显示，存在一种缺失外显子2（exon two）且引入了人工插入盒的可变剪接PARP1变体。后续的蛋白质分析证实，该敲除小鼠体内存在截短型PARP1蛋白。我们从敲除小鼠中构建了胚胎干细胞系（embryonic stem cell lines），并检测到了多聚ADP核糖（poly(ADP-ribose)，pADPr）的存在。通过蛋白质免疫印迹（Western Blotting）分析，我们在敲除胚胎干细胞中检测到pADPr水平有所下降，但免疫荧光染色（immunofluorescence staining）结果显示，敲除组与对照组胚胎干细胞的细胞核内，pADPr的分布与水平均无显著差异。此外，在Parp1与Parg双突变的胚胎干细胞中，pADPr的积累量远高于正常细胞，甚至超过低表达型Parg突变（hypomorph Parg mutant）胚胎干细胞，这提示体内存在功能活性正常的PARP1。因此，本研究结果对学界关于PARP1缺失效应的传统认知提出了挑战。为验证敲除小鼠中Parp1的mRNA表达水平，我们对对照组与敲除组小鼠的睾丸组织开展了RNA测序（RNA-seq）分析。