Establishment of a human embryonic stem cell line with homozygous TP53 R248W mutant by TALEN mediated gene editing

Genetic mutations in TP53 contribute to multiple human cancers. Here we report the generation of a H1-p53(R248W/R248W) human embryonic stem cell line harboring a homozygous TP53 R248W mutation created by TALEN-mediated precise gene editing. The H1-p53(R248W/R248W) cell line maintains a normal karyotype, robust pluripotency gene expression, and the potential to differentiate to the three germ layers.


Type of cell line
Human embryonic stem cell line

Resource utility
Growing evidence underscores the important contributions of p53 mutations in driving tumor progression. The H1-p53(R248W/R248W) line provides an ideal model for studying the functions of the p53(R248W) mutation in tumor initiation and progression in a human cell model, which opens up opportunities for development of new cancer therapeutic strategies.

Resource detail
TP53 is the most mutated gene in human cancers and leads to tumor initiation and progression in multiple cell types (Zhou et al., 2017). Previously, we established a Li Fraumeni syndrome (LFS) iPSC disease model to investigate LFS-associated osteosarcoma (Lee et al., 2015), demonstrating the potential of a pluripotent stem cell (PSC) disease model to study mutant p53 (mutp53)-associated malignancies. Generation of disease-related genetic traits in PSCs provides an attractive approach to elucidate gene function in disease development (Gingold et al., 2016). We recently generated a H1-p53(R282W/R282W) line as a laboratory resource to facilitate use of the PSC disease model in studying p53(R282W) associated malignancies (Zhou et al., 2018).
To enhance our understanding of the landscape of mutp53-associated malignancies, we generated a H1 hESC line harboring a homozygous TP53 R248W mutation. The strategy of TALEN-mediated precise gene editing is illustrated in Fig. 1A. The targeting plasmids contain (1) a pair of TALEN plasmids targeting exon7 and intron 7 of TP53, respectively; and (2) a pFNF donor vector carrying a Frt-EM7-Neo R -Frt (FNF) selection cassette flanked by 1 kb left and right homologous arms of the TP53 genomic region. H1 hESCs were transduced with these plasmids and selected by G418. G418-resistant clones were confirmed by genomic PCR using two pairs of primers (p53_5FM13 and 3FNF-N1 for left arm, 5FNF-C1 and 3p53_16821 for right arm,  bar 50 μm). Karyotype analysis confirmed the normal karyotype of the H1-p53(R248W/ R248W) line (Fig. 1I). Furthermore, PCR-based mycoplasma detection assay demonstrated that the H1-p53(R248W/R248W) line is mycoplasma-free ( Fig. 1 J). The short tandem repeat (STR) profile of H1-p53(R248W/R248W) line was identical to that of its parental H1 line (data available with authors). The characterization of the H1-p53(R248W/R248W) line was summarized in Table 1.
In summary, the H1-p53(R248W/R248W) line is karyotypically normal and retains pluripotency. This line has great potential to offer insight into the role of p53(R248W) in embryogenesis and tumorigenesis.

Cell culture
Cell culture of hESC H1 and H1-derived clones were performed as described (Zhou et al., 2018).
The left and right homologous arms were amplified from hESC H1 gDNA isolated using PureLink Genomic DNA Mini Kit (Thermo Fisher) ( Table 2). PCR products of both arms were separated on 0.8% agarose gel, extracted by QIAquick gel extraction kit (Qiagen), ligated into pGEM-Teasy vector (Promega), and confirmed by Sanger sequencing. Site-directed mutagenesis was performed to generate the R248W mutation on the left homologous arm. Both homologous arms were digested by designed restriction enzymes (EcoRI used for the right homologous arm and BamHI and NotI used for the left homologous arm) and ligated into the pFNF (Frt-EM7-Neo R -Frt) vector (Addgene plasmid #22687) to generate the TP53 R248W donor vector.

Standard PCR
Genomic DNA was isolated by Easy-DNA gDNA purification kit (

Western blotting analysis and immunofluorescent staining
Immunoblotting and immunofluorescent staining were performed as described (Zhou et al., 2018).

qRT-PCR
Total mRNA from the H1-p53(R248W/R248W) cells was extracted using TRIzol (Invitrogen). Reverse Transcription cDNA synthesis reactions were performed by iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR reactions were performed using SYBR Green PCR Master Mix (Bio-Rad) on a CFX96 machine (Bio-Rad). The reaction was performed as following parameters: 50 °C for 10 min, 95 °C for 5 min, 40 cycle of 95 °C for 10 s and 60 °C for 30 s, and 95 °C for 10 min. Samples were analyzed in triplicate and normalized to GAPDH expression.

Karyotype analysis
The G-banding karyotype was carried out in the Department of Pediatrics, Baylor College of Medicine, Texas Children's Cancer and Hematology Centers. Twenty metaphase chromosome spreads were counted and G band resolution was 400. The H1-p53(R248W/ R248W) line were karyotyped at passage 5 after the line was generated.

In vivo teratoma formation assay
Teratoma formation assay was performed as described (Zhou et al., 2018).

Mycoplasma detection
Mycoplasma detection was performed using PCR Mycoplasma Detection Kit (Applied Biological Materials Inc).

STR analysis
STR analysis was performed by the Characterized Cell Line Core Facility at the University of Texas M.D. Anderson Cancer Center. The number of STRs at 14 loci (AMEL, CSF1PO,  D13S317, D16S539, D18S51, D21S11, D3S1358, D5S818, D7S820, D8S1179, FGA,  TH01, TPOX and vWA) was assessed for H1-p53(R248W/R248W) cells. This STR profile was compared with the STR profile of parental H1 cells.  Characterization and validation.

Classification Test Result Data
Morphology Photography hESC morphology Fig. 1