Experimental Biology Center (NUBEX), University of Fortaleza (UNIFOR), Fortaleza, Ceara, Brazil.
PLoS One. 2020 Sep 18;15(9):e0239435. doi: 10.1371/journal.pone.0239435. eCollection 2020.
The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.
对基因修饰细胞进行基因分型是研究转基因和基因组编辑的关键步骤，此类编辑系统包括 CRISPR/Cas。基因组编辑事件的检测可以直接与所使用的基因分型方法相关，而该方法的成本会受到影响，因为许多实验需要分析大量样本。本研究旨在比较直接裂解法提取基因组 DNA（gDNA）提取方法在检测原代山羊细胞中的基因敲入和敲除的效果。最初，我们使用三种 gDNA 提取方案（方案 A，热变性/水冻融；方案 B，热变性/蛋白酶 K；方案 C，CellsDirect 试剂盒），使用不同数量（1000、5000 和 10000 个细胞）和两种类型的山羊原代细胞（成纤维细胞和山羊乳腺上皮细胞-GMEC）进行测试，然后通过 PCR 扩增小（GAPDH）和大扩增子（hLF 转基因）进行后续验证。所有方案均成功检测到小扩增子；然而，在 GMEC 中，只有方案 B 能有效扩增（方案 A-0%，方案 B-93%，方案 C-13.33%，P<0.05）。在一个验证性实验中，我们通过 CRISPR/Cas9 介导的缺失敲除了 GMEC 中的 TP53 基因，同时分别在成纤维细胞中敲入了含有抗 VEGF 单克隆抗体（pBC-anti-VEGF）和细菌 L-天冬酰胺酶（pBC-ASNase）的转基因。使用方案 B 和 PCR 检测成功编辑。pBC-ASNase 和 pBC-anti-VEGF 转基因的整合率分别为 93.6%和 72%，PCR 检测结果。使用 CRISPR/Cas9 对 GMEC 中的 TP53 进行双等位基因编辑的效率为 5.4%。我们的结果表明，方案 B（热变性/蛋白酶 K）可作为一种廉价且快速的方法，用于检测不同类型的原代山羊细胞中的遗传修饰，其效率与文献中使用提取试剂盒或更复杂的蛋白酶 K 制剂时描述的数值一致。
