Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
Life Sci Alliance. 2023 Mar 6;6(5). doi: 10.26508/lsa.202301944. Print 2023 May.
Here, we devised a cloning-free 3' knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5' AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (, , , and ) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested that cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereas cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepatic duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.
在这里，我们设计了一种无需克隆的斑马鱼 3' 同源敲入策略，使用 PCR 扩增的双链 DNA(dsDNA)供体，避免了靶基因的破坏。dsDNA 供体携带与内源性基因框内但通过自我切割肽与其分离的荧光蛋白和 Cre 重组酶的遗传盒。带有 5' AmC6 末端保护的引物生成了整合效率更高的 PCR 扩增子，与预先组装的 Cas9/gRNA 核糖核蛋白复合物一起注射，用于早期整合。我们靶向四个遗传位点（，，，和）并生成了 10 个敲入系，它们作为内源性基因表达的报告基因。敲入的 iCre 或 CreERT2 系用于谱系追踪，表明 细胞是多能胰腺祖细胞，逐渐限制为双潜能导管，而 细胞在肝脏和胰腺中均为多能，并逐渐限制为导管细胞。此外，在极端肝细胞丢失时，肝 导管显示出祖细胞特性。因此，我们提出了一种高效、简单的同源敲入技术，可广泛用于细胞标记和谱系追踪。
