Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia, 630090.
Novosibirsk State University, Novosibirsk, Russia, 630090.
Sci Rep. 2022 May 14;12(1):7983. doi: 10.1038/s41598-022-12071-1.
Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), β-casein (Csn2), whey acidic protein (WAP), or β-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.
转基因动物是生物技术中的一种重要工具，包括在牛奶中生产重组蛋白。传统上，表达构建体基于带有乳球蛋白特异性调节元件的杂交载体，来自α-酪蛋白（Csn1s1）、β-酪蛋白（Csn2）、乳清酸性蛋白（WAP）或β-乳球蛋白（BLG）基因。来自随机整合载体的过表达通常提供高水平的表达，但由于基因组定位不可预测，存在缺点。CRISPR-Cas9 靶向转基因整合到内源性酪蛋白基因座可以减轻对广泛的动物筛选的需求，以实现高且可重复的表达水平。我们决定评估这种“精确”的整合方法，将人粒细胞-巨噬细胞集落刺激因子（hGMCSF）基因置于小鼠内源性α-S1-酪蛋白（Csn1s1）启动子的控制下。我们设计了两种类型的转基因整合：在 Csn1s1 的第二外显子中的敲入（INS-GM）和全长 Csn1s1 替换为 hGMCSF（REP-GM），这以前从未测试过。INS-GM 方法表现出低的转基因表达和乳蛋白水平（Csn2 转录物的 0.4%；hGMCSF 为 2-11 µg/ml）。这可能是由于 hGMCSF 转基因中缺乏 3'-多聚腺苷酸化信号。REP-GM 动物表现出高的转基因表达，达到并略超过内源性 Csn1s1 的水平（Csn2 转录物的 30-40%），但产生的 hGMCSF 蛋白少于预期（0.2-0.5 mg/ml 与 Csn1s1 的 25 mg/ml 相比），表明蛋白质的翻译不是最佳的。导致 Csn1s1 敲除的纯合插入物对动物的健康没有任何长期影响。因此，在我们的实验设计中，与过表达方法相比，特异性转基因整合到酪蛋白基因座没有提供任何显著优势。
