Adapting Mouse Genome Editing Technique from Scratch Using in utero Electroporation

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Mouse genome modification requires costly equipment and highly skilled personnel to manipulate zygotes. A number of zygote electroporation techniques were reported to be highly efficient in gene delivery. One of these methods called i-GONAD (improved Genome-editing via Oviductal Nucleic Acids Delivery) describes electroporation-based gene transfer to zygotes in utero. Here we adopted this technology to develop an easy-to-use and cost-effective pipeline enabling mouse genome-editing from scratch with minimal requirements to operator skills and animal use. We chose the CRISPR/Cas9 system as a genome editing tool and i-GONAD as a gene delivery method to produce Il10 knockout in C57BL/6 mice. Three animals out of 13 delivered pups (23%) were genetically compromised at Il10 locus suggesting the feasibility of the approach. This protocol provides detailed description of the used technical settings paired with troubleshooting tips and could be of interest to those who aim at establishing in-house mouse transgenesis pipeline at minimal equipment cost from scratch.

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作者简介

Yu. Popova

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk State Agrarian University

Email: kozhevnikova@mcb.nsc.ru
俄罗斯联邦, Novosibirsk, 630090; Novosibirsk, 630039

V. Bets

Novosibirsk State Technical University

Email: kozhevnikova@mcb.nsc.ru
俄罗斯联邦, Novosibirsk, 630073

E. Omelina

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences

Email: kozhevnikova@mcb.nsc.ru
俄罗斯联邦, Novosibirsk, 630090

L. Boldyreva

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences; Scientific Research Institute of Neurosciences and Medicine, Novosibirsk, 630117

Email: kozhevnikova@mcb.nsc.ru
俄罗斯联邦, Novosibirsk, 630090; Novosibirsk, 630117

E. Kozhevnikova

Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk State Agrarian University

编辑信件的主要联系方式.
Email: kozhevnikova@mcb.nsc.ru
俄罗斯联邦, Novosibirsk, 630090; Novosibirsk, 630039

参考

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3. Fig. 1. The scheme of CRISPR/Cas9 genome editing in combination with the i-GONAD method. a – Synthesis of sgRNA (single guide RNA). b – The i-GONAD method (improved Genome-editing via Oviductal Nucleic Acids Delivery). b – Suturing and postoperative procedures. The drawing was created on the website biorender.com.

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4. Fig. 2. Adaptation and testing of the i-GONAD method. a – Testing of sdRNA in vitro. Electrophoresis in agarose gel of a ScaI-linearized pBluescript vector SK (+) carrying a fragment of the genomic region of exon 1 or exon 2 of the Il10 gene. RNP – ribonuclear complexes of cdRNA and Cas9 protein. b – Survival of zygotes in vitro after electroporation in utero. EP – electroporation. b – In utero electroporation efficiency test using 4kDa FITC-dextran (FD4).

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5. Fig. 3. Genotyping of offspring. a is a schematic illustration of the wild–type Il10 locus. Exons (from x1 to x5) and introns are indicated by black rectangles and a gray line, respectively. The two target sequences (ex1 and ex2) are underlined and shown in blue. The sequences of the motif adjacent to the protospacer (PAM) are shown in red. b – Direct Sanger sequencing of the genomic regions of the Il10 gene in F1 progeny. Transgene #58 has abnormalities in exon 1, the other two (#27 and #32) in exon 2 of the Il10 gene. b – Comparison of the amino acid sequences of the wild-type Il10 gene and offspring after i-GONAD. The control amino acid sequence of the wild-type Il10 protein is shown above. The amino acid sequences of the mutated Il10 proteins (derived from transgenes #58, #27 and #32) are shown below. The predicted consequences of mutation of amino acid sequences are highlighted in red. The stop codons are marked with *.

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