Inducible Avp Knockout Mouse Line

Arginine vasopressin (AVP) is a peptide hormone coded by the Avp gene, synthesized in the hypothalamus and secreted by the posterior pituitary. Dysregulation of AVP secretion contributes to a variety of human diseases. Previous studies of functional roles of AVP have been largely dependent on the use of Brattleboro rats, which manifest a spontaneous mutation in the Avp gene and lack circulating AVP. Despite their utility, Brattleboro rats are difficult to breed owing largely to the fixed nature of the Avp mutation, resulting in increased neonatal death and behavioral effects in the adults. Consequently, commercial breeders have ceased production, despite a continued need. Therefore, the main goal of this project is to create an effective experimental Avp knockout mouse model that could be used in renal and neuroendocrine research to study the control of water balance by AVP. We employed CRISPR/Cas9 to flox a portion of exon 2 of the Avp gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Mice harboring the floxed allele were mated to B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J mice that globally express a tamoxifen-inducible Cre recombinase. The resultant inducible Avp knockout mice (Cre+Avpflx/flx) show no signs of polydipsia or polyuria prior to induction, indicating that the floxed gene maintains its wild-type function. The administration of an exogenous inducer like tamoxifen to (8-10) week-old mice, induced Cre-mediated recombination that resulted in a decrease in urine osmolality from 2076 ± 138 to 122 ± 6 mOsm/kgH2O on day 31 after induction. Sanger sequencing demonstrated the expected 1245 bp deletion at the Avp locus. Immunoblotting of AQP2 in the inner medulla showed a significant decrease in AQP2 band density in (Cre+Avpflx/flx) mice to 27 ±1 4 % of values in Cre- floxed control mice. This inducible Avp knockout mouse model provides researchers with a valuable tool to investigate the consequences of Avp gene deletion in a controlled and inducible manner. Overall design: The process begins with the design (CHOPCHOP) and synthesis of guide RNAs (gRNAs) that target the desired regions (first intron and 3' downstream) within the vasopressin gene. These gRNAs, when complexed with the Cas9 nuclease, induce site-specific double-strand breaks (DSBs) at the AVP locus. To introduce the loxP sites, a DNA repair template containing the loxP sequences and restriction enzyme sites (BamHI and EcorI) are provided along with the CRISPR/Cas9 components. The repair template serves as a template for homology-directed repair (HDR) and facilitates the precise insertion of loxP sequences flanking the vasopressin gene. Successful insertion of the two loxP sites was confirmed by PCR using primers flanking the targeted regions. Restriction enzymes were used to confirm correct targeting. Additionally, PCR amplification of DNA sequences spanning the two loxP sites was carried out, followed by cloning into the TA-cloning vector and subsequent sanger sequencing for further confirmation. Sequencing analysis of the founder mice demonstrated precise insertion of the loxP sites into each intended location within the Avp locus, without introducing mutations at the loxP sites.精氨酸加压素（Arginine vasopressin, AVP）是由Avp基因编码的肽类激素，于下丘脑合成、垂体后叶分泌。AVP分泌失调可引发多种人类疾病。既往针对AVP功能的研究大多依赖布拉特尔伯勒大鼠（Brattleboro rats），该品系大鼠的Avp基因存在自发突变，无法分泌循环AVP。尽管该品系大鼠具有科研应用价值，但由于Avp突变固定，其繁育难度极大，会导致新生仔鼠死亡率升高，还会使成年个体出现行为学异常。因此，尽管科研界仍有需求，商业繁育机构已停止该品系大鼠的繁育工作。 基于此，本项目的核心目标是构建一款高效的Avp基因敲除小鼠实验模型，用于肾脏及神经内分泌领域研究，以探究AVP对机体水平衡的调控机制。我们采用CRISPR/Cas9系统对Avp基因第2外显子的部分片段进行了loxP位点侧翼锚定操作。通过靶向区域侧翼引物进行PCR扩增，证实了两个loxP位点已成功插入。将携带floxed等位基因的小鼠与B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J品系小鼠进行交配，该品系小鼠可全局表达他莫昔芬诱导型Cre重组酶。最终获得的诱导型Avp基因敲除小鼠（Cre+Avpflx/flx）在诱导处理前无多饮、多尿症状，表明该floxed基因仍维持野生型功能。 向8~10周龄的小鼠施加他莫昔芬等外源性诱导剂后，可诱导Cre介导的同源重组，在诱导后第31天时，小鼠尿渗透压从2076±138毫渗摩尔每千克水降至122±6毫渗摩尔每千克水。桑格测序（Sanger sequencing）证实，Avp基因位点发生了预期的1245 bp片段缺失。对肾内髓质的水通道蛋白2（Aquaporin 2, AQP2）进行免疫印迹分析，结果显示，Cre+Avpflx/flx小鼠的AQP2条带密度显著降低，仅为Cre- floxed对照小鼠的27±14%。该诱导型Avp基因敲除小鼠模型为研究者提供了一款宝贵工具，可用于在可控、可诱导的条件下探究Avp基因缺失所产生的生物学效应。 实验整体设计如下： 首先通过CHOPCHOP工具设计并合成靶向加压素基因目的区域（第一内含子及3'下游序列）的向导RNA（guide RNAs, gRNAs）。上述gRNA与Cas9核酸酶形成复合物后，可在AVP基因位点诱导位点特异性双链断裂（double-strand breaks, DSBs）。为插入loxP位点，我们向CRISPR/Cas9系统中加入了包含loxP序列及限制性内切酶位点（BamHI与EcoRI）的DNA修复模板。该修复模板可作为同源定向修复（homology-directed repair, HDR）的模板，帮助精准将loxP序列整合至加压素基因的侧翼区域。通过靶向区域侧翼引物进行PCR扩增，可证实两个loxP位点已成功插入。通过限制性内切酶酶切验证了靶向整合的准确性。此外，我们对跨越两个loxP位点的DNA序列进行了PCR扩增，将产物克隆至TA克隆载体后进行桑格测序（Sanger sequencing），以进一步验证整合结果。对首建小鼠的测序分析显示，loxP位点已精准插入Avp基因位点的预定位置，且loxP位点区域未引入任何突变。