Identification of the Transgene Integration Site and Host Genome Changes in MRP8-Cre/ires-EGFP Transgenic Mice by Targeted Locus Amplification.
Identification of the Transgene Integration Site and Host Genome Changes in MRP8-Cre/ires-EGFP Transgenic Mice by Targeted Locus Amplification.
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DOI：
10.3389/fimmu.2022.875991
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发表时间：
2022
影响因子：
7.3
通讯作者：
Luo HR
中科院分区：
文献类型：
--
作者：
Wang G;Zhang C;Kambara H;Dambrot C;Xie X;Zhao L;Xu R;Oneglia A;Liu F;Luo HR
MRP 8-Cre-ires/EGFP转基因小鼠（Mrp 8 creTg，C57 BL/6 J遗传背景）因在中性粒细胞中特异性表达Cre重组酶和EGFP报告基因而在免疫学和血液学研究中受到欢迎。它经常与携带loxP侧翼基因的其他转基因系杂交，以实现中性粒细胞中的限制性基因敲除。然而，由于建立该品系的方式，关于宿主基因组中的MRP 8-Cre-ires/EGFP转基因的基本知识，例如其整合位点和侧翼序列，在很大程度上仍然是未知的，这阻碍了稳健的实验设计和数据解释。在这里，我们使用了最近开发的技术，靶向基因座扩增（TLA）测序，以填补这些知识空白。我们发现MRP 8-Cre-ires/EGFP转基因整合到宿主小鼠基因组的5号染色体（5 qG 2）中。这种整合导致宿主基因组序列的44 kb缺失，导致Serpine 1的完全缺失和Ap 1 s1的部分缺失。在确定了转基因的侧翼序列后，我们设计了一种新的基因分型方案，可以区分纯合、杂合和野生型Mrp 8 creTg小鼠。令我们惊讶的是，杂交杂合子小鼠没有产生纯合子Mrp 8 creTg小鼠，最有可能是由于产前致死性破坏Ap 1 s1基因表达。
The MRP8-Cre-ires/EGFP transgenic mouse (Mrp8creTg, on C57BL/6J genetic background) is popular in immunological and hematological research for specifically expressing Cre recombinase and an EGFP reporter in neutrophils. It is often crossed with other transgenic lines carrying loxP-flanked genes to achieve restricted gene knockout in neutrophils. However, due to the way in which the line was created, basic knowledge about the MRP8-Cre-ires/EGFP transgene in the host genome, such as its integration site(s) and flanking sequences, remains largely unknown, hampering robust experimental design and data interpretation. Here we used a recently developed technique, targeted locus amplification (TLA) sequencing, to fill these knowledge gaps. We found that the MRP8-Cre-ires/EGFP transgene was integrated into chromosome 5 (5qG2) of the host mouse genome. This integration led to a 44 kb deletion of the host genomic sequence, resulting in complete deletion of Serpine1 and partial deletion of Ap1s1. Having determined the flanking sequences of the transgene, we designed a new genotyping protocol that can distinguish homozygous, heterozygous, and wildtype Mrp8creTg mice. To our surprise, crossing heterozygous mice produced no homozygous Mrp8creTg mice, most likely due to prenatal lethality resulting from disrupted Ap1s1 gene expression.
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