Lab Anim Res.  2018 Dec;34(4):264-269. 10.5625/lar.2018.34.4.264.

Generation of knockout mouse models of cyclin-dependent kinase inhibitors by engineered nuclease-mediated genome editing

Affiliations
  • 1Department of Biochemistry, College of Life Science & Biotechnology, Yonsei University, Seoul, Korea. hwl@yonsei.ac.kr jhlee13@gmail.com rohjaeil@gmail.com

Abstract

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors p16(Ink4a) (Cdkn2a, cyclin-dependent kinase inhibitor 2a), p19(Arf) (an alternative reading frame product of Cdkn2a,), and p27(Kip1) (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The p16(Ink4a) and p19(Arf) knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27(Kip1) knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

Keyword

TALEN; CRISPR/Cas9; cyclin-dependent kinase inhibitor

MeSH Terms

Animals
Cell Cycle
Codon, Nonsense
Cyclin-Dependent Kinase Inhibitor p16
DNA
Exons
G1 Phase
Genome*
Melanoma
Mice
Mice, Knockout*
Mutagenesis, Insertional
Neurodegenerative Diseases
Phosphotransferases*
Reading Frames
Sarcoma
Codon, Nonsense
Cyclin-Dependent Kinase Inhibitor p16
DNA
Phosphotransferases

Figure

  • Figure 1 TALEN-mediated generation of a p16Ink4a knockout mouse model. (A and B) Representative views of the transcription activator-like effector nuclease (TALEN) targeting strategy for generation of p16Ink4a KO mice in both FVB (A) and B6 (B) strains. A schematic of the Cdkn2a locus with TALEN-binding nucleotides (blue) and the amino acid sequences (purple) of the WT (upper) and KO (lower) mice are shown. p16Ink4a exons are indicated with boxes and introns are indicated by lines. Red asterisks indicate stop codons. (C and D) Representative results of PCR genotyping for FVB (C) and C57BL/6 (D) strains of WT and p16Ink4a KO mice. +/+, WT; +/−, Heterozygote KO; −/−, Homozygote KO. (E and F) Western blot analyses for p16 in FVB (E) and B6 (F) mouse embryonic fibroblasts. +/+ indicates WT and −/− indicates the p16Ink4a KO.

  • Figure 2 TALEN-mediated generation of a p19Arf knockout mouse model. (A and B) Representative views of the transcription activator-like effector nuclease (TALEN) targeting strategy for generation of p19Arf KO mice in both FVB (A) and B6 (B) strains. A schematic of the Cdkn2a locus with TALEN-binding nucleotides (blue) and the amino acid sequences (purple) of the WT (upper) and KO (lower) mice are shown. p19Arf exons are indicated with boxes and introns are indicated by lines. Red asterisks indicate stop codons. (C and D) Representative results of PCR genotyping for FVB (C) and B6 (D) strains of WT and p19Arf KO mice. +/+, WT; +/−, Heterozygote KO; −/−, Homozygote KO.

  • Figure 3 CRISPR/Cas9-mediated generation of B6-p27Kip1 knockout mouse model. (A) Representative view of the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) targeting strategy for generation of p27Kip1 KO mouse. A schematic of the Cdkn1b locus with single guide RNA target nucleotides (blue) and the amino acid sequences (purple) of the wild-type (upper) and knockout (lower) mice are shown. Exons are indicated with boxes and introns are indicated by lines. Red asterisks indicate stop codons. The protospacer adjacent motif for Cas9 is indicated in red. (B) A representative result of PCR genotyping for B6 strain of WT and p27Kip1 KO mouse. +/+, WT; +/−, Heterozygote KO. (C) Semi-quantitative PCR analysis for p27 transcript in the liver of WT and p27Kip1 KO mouse. +/+, WT; −/−, Homozygote p27Kip1 KO.


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