【原】举几个例子，看别人如何构建Knock in(KI)细胞系

GCTA 2022-06-11 发布于贵州

展开全文

HACS

1. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine -casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination

原文摘要：

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland(腺), and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine (牛， [ˈboʊvaɪn] )-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the -casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine -casein gene.

Figure 1 The knock-in strategy in the bovine -casein gene locus. The diagram shows homologous recombination of the knockin vector in the bovine -casein gene locus. The PGKneo gene and DT-A gene without the polyA signal sequence were used as positive and negative selection markers, respectively. The PCR primer pairs used for detecting homologous recombination events are shown by name.

左边（5’端）是5.98kb的同源臂，右边（3’端）是2.08kb的同源臂，中间带有目的基因和Neo抗性基因（用于筛选）。

此外，他们还设计了一个报告系统评估CRISPR/Cas9的工作情况：（就是为了水一张Figure吧）

参考文献： Zygote 24 (June), pp. 442–456. c Cambridge University Press 2015 doi:10.1017/S0967199415000374 First Published Online 22 July 2015

2. Highly efficient generation of knock-in transgenic medaka（青鳉） by CRISPR/Cas9- mediated genome engineering

原文摘要：

Background: Medaka (Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.

Results: We report the highly efficient generation of knock-in transgenic medaka vianon-homologous[hoʊˈmɑːləɡəs] end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.

Conclusion: With its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.

Keywords: Medaka, Transgenic, CRISPR/Cas9, Knock-in

Fig. 1 Strategy for the generation of knock-in medaka and generation of Tg[vacht-hs:lRl-GFP] strains. (a) For the generation of knock-in transgenic fish, sgRNA1 (for genome digestion), sgRNA2 (for plasmid digestion), donor plasmid with a bait sequence, and Cas9 mRNA are co-injected into one-cell-stage medaka embryos. (b) A schematic representation of the vacht locus (grey box) and the sgRNA target sites (orange box), and the reporter gene construct consisting of the Tbait (brown box), medaka hsp70 promoter (hsP, blue box), loxP, RFP-pA (red box), loxP, and GFP-pA (green box). After injection, the concurrent cleavage of the targeted genomic locus and the Tbait-hs-lRl-GFP reporter plasmid results in the integration of the reporter by non-homologous end joining (NHEJ). The scheme shows the forward integration of the reporter.(红细线表示切割位点)

这个是非同源末端修复，无需同源臂。sgRNA1和sgRNA2内部各切一刀后断口就可以连接起来。理论上自连的概率极高，但是他们说sgRNA2的断口有30%左右的概率可以和sgRNA1的断口连接。但这是青鳉里面才能发生，其他模式动物细胞应该不行。

参考文献： Watakabe et al. Zoological Letters (2018) 4:3 DOI 10.1186/s40851-017-0086-3

3. CRISPR/Cas9介导的外源基因靶向插入鸡EAV-HP基因组

原文摘要：

本研究旨在通过CRISPR/Cas9介导外源基因靶向插入鸡EAV-HP基因组。首先设计特异性引物并扩增鸡内源性病毒(EAV-HP)左右同源臂和增强型绿色荧光蛋白(eGFP)基因表达盒，然后通过重叠延伸PCR技术将两个同源臂DNA连接至eGFP表达盒两侧，获得全长DNA片段LER，并克隆至pMD19-T载体，获得携带eGFP基因的供体载体pMDT-LER。随后在HEK293T细胞中验证供体载体pMDT-LER能成功表达eGFP后，将EAV-HP打靶载体和供体载体共转染至DF-1细胞，观察绿色荧光阳性细胞，提取细胞基因组，PCR检测外源基因eGFP成功整合至鸡基因组EAV-HP位点。最后，将转基因细胞DF-1传至第7代，用PCR和Western blotting检测eGFP在转基因细胞中稳定表达。文中初步验证外源基因eGFP能整合至鸡EAV-HP位点并稳定表达，为转基因鸡的研究提供新整合位点。

关键词：CRISPR/Cas9 鸡内源性病毒 EAV-HP 整合位点供体载体

图 1 CRISPR/Cas9介导外源基因eGFP插入鸡EAV-HP位点示意图(A：供体载体结构示意图；B：供体载体整合至鸡基因组原理示意图)Fig. 1 Schematic diagram of CRISPR/Cas9-mediated foreign gene targeted knock-in into the chicken genomic EAV-HP locus.(A) Schematic diagram of donor vector. (B) Schematic diagram of donor vector integration

供体载体构建及鉴定

本研究构建的供体载体含 eGFP 表达盒，其两边携带EAV-HP 同源序列。首先利用 PCR 扩增EAV-HP 的左右同源臂以及 eGFP 表达盒，然后通过重叠延伸 PCR 将 3 个 DNA 片段连接，其 PCR产物简称 LER。最后利用 TA 克隆将 LER 片段插入 T 载体 pMD19-T，经菌落 PCR、酶切鉴定和DNA 测序，证明 LER 片段成功克隆至 T 载体，且无突变，获得重组载体 pMDT-LER (图 1A)。该重组载体将为 CRISPR/Cas9 介导 eGFP 基因靶向插入鸡 EAV-HP 位点提供供体 DNA (图 1B)。

参考文献：