Department of Translational Molecular Pathology and Sheikh Ahmed Pancreatic Cancer Research Center, UT MD Anderson Cancer Center, Houston, TX, USA.
Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA.
Lab Invest. 2019 Jul;99(8):1233-1244. doi: 10.1038/s41374-018-0171-z. Epub 2019 Feb 6.
Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single "workhorse" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic Kras allele at the endogenous locus, and insertion deletion ("indel") and frameshift mutations of targeted tumor suppressor alleles. By using a single "workhorse" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.
基因工程小鼠模型（GEMMs）再现了胰腺导管腺癌（PDAC）中的主要遗传驱动因素，为这种致命肿瘤的发病机制提供了前所未有的见解。尽管如此，生成自发模型是一个昂贵、耗时和劳动密集的过程，特别是当涉及组织特异性表达或复合等位基因缺失时。此外，目前许多 PDAC GEMMs 导致胚胎期、全胰腺激活或驱动等位基因缺失，这两者都与对应人类疾病的情况不符。CRISPR/Cas9 基因编辑的出现可能会规避传统繁殖方案的许多上述缺点，但确保体内基因编辑的效率仍然是一个挑战。在这里，我们使用一种在成年胰腺中由 p48 启动子驱动表达 Cas9 的“主力”小鼠品系，开发了一种高效生成具有复杂基因型的 PDAC GEMMs 的流水线。通过腺相关病毒（AAV）介导的将多聚体向导 RNA（sgRNA）递送到 p48-Cre；LSL-Cas9 小鼠的成年鼠胰腺中，我们证实了我们能够通过同源定向修复（HDR）表达致癌性 Kras 等位基因，同时通过 CRISPR 诱导破坏合作等位基因（Trp53、Lkb1 和 Arid1A）。所得 GEMMs 显示出一系列前体病变（胰腺上皮内瘤变[PanIN]或导管内乳头状黏液性肿瘤[IPMN]，最终进展为 PDAC）。对所得小鼠 PDAC 的下一代测序证实了内源性位点致癌性 Kras 等位基因的 HDR，以及靶向肿瘤抑制基因等位基因的插入缺失（“indel”）和移码突变。通过使用单一的“主力”小鼠品系和用于体内基因编辑的最佳 AAV 血清型以及组合驱动基因，我们为 PDAC 基因组的研究提供了一个简单的自发平台。
