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 p16Ink4a (Cdkn2a, cyclin-dependent kinase inhibitor 2a), p19Arf (an alternative reading frame product of Cdkn2a,), and p27Kip1 (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 p16Ink4a and p19Arf knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27Kip1 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.
Highlights
Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer
Cdkn2a is a cell cycle regulating gene encoding alternate proteins, p16Ink4a and p19Arf, that arrest the G1 phase through CDK4/6 inhibition [10,12]. As they are tumor suppressors, p16Ink4a and p19Arf can be targeted for treatment of various cancers, including melanoma, cervical, and esophageal cancers [4,13,22, 30,32]. p27Kip1 is another type of cyclin-dependent kinases (CDKs) inhibitor which suppresses CDK2, a CDK required for S phase entry [23]
transcription activator-like effector nucleases (TALENs) consist of transcription activator-like (TAL) effectors fused with a FokI cleavage domain [9]
Summary
Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. The p16Ink4a and p19Arf knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27Kip knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas). The p16Ink4a and p19Arf knockout mice were generated via transcription activator-like effector nucleases (TALENs), and p27Kip 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. Cdkn2a (cyclindependent kinase inhibitor 2a) is a cell cycle regulating gene encoding alternate proteins, p16Ink4a and p19Arf, that arrest the G1 phase through CDK4/6 inhibition [10,12] As they are tumor suppressors, p16Ink4a and p19Arf can be targeted for treatment of various cancers, including melanoma, cervical, and esophageal cancers [4,13,22, 30,32]. CRISPR/Cas is a ribonucleoprotein complex, containing a single guide RNA (sgRNA) with a targeting sequence and a Cas protein which recognizes a protospacer adjacent motif and cleaves the target site [24]
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