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Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting

Jeong, Yeon-Ju, Cho, Jeongin, Kwak, Jina, Sung, Young Hoon, Kang, Byeong-Cheol·2022·Animal Cells and Systems
DOI 10.1080/19768354.2022.2151509PMC PMC9809370
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Figure 1. Cas9-mediated p53 gene targeting in primary skin fibroblasts isolated from a common marmoset monkey. (A) Schematic representation of the p53-specific sgRNA on exon 7. Target and PAM sequences are denoted by red and blue colors, respectively. Dotted boxes indicate the amino acids critical for the DNA-binding function of p53 protein as a transcription factor. (B) Endonuclease activity induced by p53-specific sgRNA (sgp53). After infection with lentivirus expressing both Cas9 and p53-specific sgRNA, Cas9-induced indel mutations was examined using genomic DNA samples from parental primary skin fibroblasts (−) and puromycin-selected cells (+) by T7E1 assay. (C) Mutated p53 sequences observed in the mutant cell clones. − denotes deleted nucleotides; sequences in lower case denote nucleotide insertions. (D) Western blot analysis of p53 proteins in primary skin fibroblasts (parental; Pa) and p53-/- skin fibroblasts (clone #4 in c).
Figure 1. Cas9-mediated p53 gene targeting in primary skin fibroblasts isolated from a common marmoset monkey. (A) Schematic representation of the p53-specific sgRNA on exon 7. Target and PAM sequences are denoted by red and blue colors, respectively. Dotted boxes indicate the amino acids critical for the DNA-binding function of p53 protein as a transcription factor. (B) Endonuclease activity induced by p53-specific sgRNA (sgp53). After infection with lentivirus expressing both Cas9 and p53-specific sgRNA, Cas9-induced indel mutations was examined using genomic DNA samples from parental primary skin fibroblasts (−) and puromycin-selected cells (+) by T7E1 assay. (C) Mutated p53 sequences observed in the mutant cell clones. − denotes deleted nucleotides; sequences in lower case denote nucleotide insertions. (D) Western blot analysis of p53 proteins in primary skin fibroblasts (parental; Pa) and p53-/- skin fibroblasts (clone #4 in c).CC BY · PubMed Central
Figure 2. Generation of mutant marmoset skin fibroblasts deficient in both p16INK4A and p14ARF genes using CRISPR-Cas9. (A) Schematic representation of CDKN2A-specific sgRNA on exon 2, which is common to both p16INK4A and p14ARF. Target and PAM sequences are denoted by red and blue colors, respectively. (B) Endonuclease activity of Cas9 induced by common exon 2-specific sgRNA (sgCDKN2A). T7E1 assays were conducted using genomic DNA samples from parental primary skin fibroblasts (−) and puromycin-selected cells (+) after infection with lentivirus expressing both Cas9 and sgCDKN2A. (C) Mutated CDKN2A sequences observed in the clones. A polymorphic nucleotide is shaded in gray. Ref, reference genomic DNA sequence of the CDKN2A locus; Pa, parental marmoset skin fibroblasts; − denotes deleted nucleotides; sequences in lower case denote nucleotide insertions. (D) Western blot analysis of p16INK4A and p14ARF proteins in p53-/- cells (Figure 1 clone #4) and CDKN2A-/- cells (clone #1 in c). Short exp., short exposure time; long exp., long exposure time.
Figure 2. Generation of mutant marmoset skin fibroblasts deficient in both p16INK4A and p14ARF genes using CRISPR-Cas9. (A) Schematic representation of CDKN2A-specific sgRNA on exon 2, which is common to both p16INK4A and p14ARF. Target and PAM sequences are denoted by red and blue colors, respectively. (B) Endonuclease activity of Cas9 induced by common exon 2-specific sgRNA (sgCDKN2A). T7E1 assays were conducted using genomic DNA samples from parental primary skin fibroblasts (−) and puromycin-selected cells (+) after infection with lentivirus expressing both Cas9 and sgCDKN2A. (C) Mutated CDKN2A sequences observed in the clones. A polymorphic nucleotide is shaded in gray. Ref, reference genomic DNA sequence of the CDKN2A locus; Pa, parental marmoset skin fibroblasts; − denotes deleted nucleotides; sequences in lower case denote nucleotide insertions. (D) Western blot analysis of p16INK4A and p14ARF proteins in p53-/- cells (Figure 1 clone #4) and CDKN2A-/- cells (clone #1 in c). Short exp., short exposure time; long exp., long exposure time.CC BY · PubMed Central
Figure 3. Immortalized phenotypes observed in both p53- and CDKN2A-deficient marmoset skin fibroblasts. (A, B) Cumulative population doublings (PDs) of p53-deficient (A) and CDKN2A-deficient (B) marmoset skin fibroblasts. Growth of polyclonal cells (purple triangles) and monoclonal mutant cells (red rectangles; p53-/- clone #4 and CDKN2A-/- clone #1) was compared with that of parental primary skin fibroblasts (blue circle). (C) Senescence-associated β-galactosidase (SAβ-gal) assays conducted with marmoset skin fibroblasts and immortalized p53- and CDKN2A-deficient clones at the denoted passages. Scale bar, 200 μm.
Figure 3. Immortalized phenotypes observed in both p53- and CDKN2A-deficient marmoset skin fibroblasts. (A, B) Cumulative population doublings (PDs) of p53-deficient (A) and CDKN2A-deficient (B) marmoset skin fibroblasts. Growth of polyclonal cells (purple triangles) and monoclonal mutant cells (red rectangles; p53-/- clone #4 and CDKN2A-/- clone #1) was compared with that of parental primary skin fibroblasts (blue circle). (C) Senescence-associated β-galactosidase (SAβ-gal) assays conducted with marmoset skin fibroblasts and immortalized p53- and CDKN2A-deficient clones at the denoted passages. Scale bar, 200 μm.CC BY · PubMed Central
Figure 4. Induction of p53 target genes by genotoxic stresses in immortalized CDKN2A-deficient marmoset skin fibroblasts. (A) Western blot analysis of p53 and its target gene products, MDM2 and p21WAF1, in immortalized p53- and CDKN2A-deficient marmoset skin fibroblasts treated with 200 ng/ml adriamycin for 4 h. (B) Assessment of adriamycin-induced p53 target gene (MDM2 and p21WAF1) transcription by semi-quantitative RT-PCR. Immortalized marmoset cells were treated as in a. The asterisk indicates truncated marmoset p53 proteins. (C) Western blot analysis of p53 and its target gene products, MDM2 and p21WAF1, in immortalized p53- and CDKN2A-deficient marmoset skin fibroblasts treated with 0.5 μM etoposide for 4 h. The asterisk indicates truncated marmoset p53 proteins. (D) Etoposide induction of p53 target gene (MDM2 and p21WAF1) transcription in immortalized marmoset cells. Semi-quantitative RT-PCR reactions were conducted with cells treated as in c.
Figure 4. Induction of p53 target genes by genotoxic stresses in immortalized CDKN2A-deficient marmoset skin fibroblasts. (A) Western blot analysis of p53 and its target gene products, MDM2 and p21WAF1, in immortalized p53- and CDKN2A-deficient marmoset skin fibroblasts treated with 200 ng/ml adriamycin for 4 h. (B) Assessment of adriamycin-induced p53 target gene (MDM2 and p21WAF1) transcription by semi-quantitative RT-PCR. Immortalized marmoset cells were treated as in a. The asterisk indicates truncated marmoset p53 proteins. (C) Western blot analysis of p53 and its target gene products, MDM2 and p21WAF1, in immortalized p53- and CDKN2A-deficient marmoset skin fibroblasts treated with 0.5 μM etoposide for 4 h. The asterisk indicates truncated marmoset p53 proteins. (D) Etoposide induction of p53 target gene (MDM2 and p21WAF1) transcription in immortalized marmoset cells. Semi-quantitative RT-PCR reactions were conducted with cells treated as in c.CC BY · PubMed Central

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Cited by

Metadata

Authors
Jeong, Y.-J., Cho, J., Kwak, J., Sung, Y. H., & Kang, B.-C.
Affiliations
  • Convergence Medicine Research Center, Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea
  • Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
ORCID
Sung, Young Hoon
Venue
Animal Cells and Systems
Publisher
Informa UK Limited
Published
2022-11-02
Volume
26
Issue
6
Pages
266-274
ISSN
  • 1976-8354
  • 2151-2485
DOI
10.1080/19768354.2022.2151509
PMC
PMC9809370
Cited by
11
References
4
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • National Research Foundation of Korea
  • Ministry of Science, ICT, and Future Planning (2021IP0050)
  • Asan Institute for Life Sciences, Asan Medical Center

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