Engineering a humanized telomerase reverse transcriptase gene in mouse embryonic stem cells

Abstract

Telomerase is expressed in adult mouse, but not in most human, tissues and mouse telomeres are much longer than those in humans. This interspecies difference of telomere homeostasis poses a challenge in modeling human diseases using laboratory mice. Using chromatinized bacterial artificial chromosome reporters, we discovered that the 5' intergenic region, introns 2 and 6 of human telomerase gene (hTERT) were critical for regulating its promoter in somatic cells. Accordingly, we engineered a humanized gene, hmTert, by knocking-in a 47-kilobase hybrid fragment containing these human non-coding sequences into the mTert locus in mouse embryonic stem cells (mESCs). The hmTert gene, encoding the wildtype mTert protein, was fully functional, as a mESC line with homozygous hmTert alleles proliferated for over 400 population doublings without exhibiting chromosomal abnormalities. Like human ESCs, the engineered mESCs contained high telomerase activity, which was repressed upon their differentiation into fibroblast-like cells in a histone deacetylase-dependent manner. Fibroblast-like cells differentiated from these mESCs contained little telomerase activity. Thus, telomerase in mESCs with the hmTert alleles was subjected to human-like regulation. Our study revealed a novel approach to engineer a humanized telomerase gene in mice, achieving a milestone in creating a mouse model with humanized telomere homeostasis.

Figure 1

The involvement of distal genomic sequences in the repression of hTERT promoter in human fibroblasts. A schematic diagram of all BAC reporters is shown at the top. Shown below are illustrations of H(wt), M(wt), and chimera BACs. Black and light grey lines represent human and mouse genomic sequences, respectively. (A) BAC reporters made from human and mouse genomic sequences, H(wt) and M(wt), respectively. (B) H(mPro) and M(hPro), in which TERT promoter regions were swapped between H(wt) and M(wt). (C) M(h5IR), M(h5IRI2) and M(h5IRI2I6), 5IR, I2, and I6 represent 5′ intergenic region, introns 2 and 6 of the hTERT gene, replacing their corresponding mouse sequences in M(hPro). Bar graphs, activities of TERT promoters in chromatinized BAC reporters in Tel⁻ (−) and Tel⁺ (+) human fibroblast cells. Luciferase activities were measured in 96-well plates, treated without (black bars) or with TSA (white bars). The TERT promoter activities were measured as the ratios of Firefly to Renilla luciferase activities. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

TERT promoter activities from chromatinized BAC reporter in mouse ESCs and their differentiated derivatives. (A) TERT promoter activities in differentiating EB cultures. ESCs containing BAC reporters were cultured in the absence of feeder cells and LIF to form EBs. Luciferase activities were measured in 3, 6, 10, and 14 days, and normalized to those of the undifferentiated ESCs. (B) TERT promoter activities in differentiated fibroblast-like cultures. The promoter activities were normalized to those of ESCs. *p < 0.05; ***p < 0.001, compared to their respective activities in ESCs.

Figure 3

Engineering of an hmTert allele in mouse ESCs. (A) A diagram of the targeting BAC. Black and grey portions of horizontal lines indicate human and mouse genomic sequences, respectively. Vertical bars show relative positions and sizes of exons of the CRR9 (left) and TERT (right) genes. TK and Puro are thymidine kinase and puromycin-resistant markers, respectively. The targeting BAC along with two pX458-sgRNAs targeting mouse sequences near the boundaries of homologous recombination (indicated by flash signs) were cotransfected into ESCs and puromycin/GCV-resistant colonies were selected. The Puro marker was subsequently removed following transient transfection of pCBM, a Cre-expressing plasmid. The knock-in of a 47-kb genomic region as the result of homologous recombination is indicated. A small white dot at the 3′ end of Tert exon 2 indicates the silent mutations that distinguish mRNAs from mTert and hmTert loci in RT-qPCR assays. (B) mRNA expression from the hmTert locus. Total RNAs from ESCs containing mTert and/or hmTert loci were subjected to RT-qPCR analysis using probes that distinguish mTert and hmTert mRNAs, and the data were normalized to 18S rRNA. a-d were four independent m/hm clones. (C) Telomerase activity in ESCs with mTert and/or hmTert alleles. Telomerase activities in extracts of 2000 or 20 cells were measured using TRAP assay. m/m, wildtype ESCs containing two mTert alleles; hm/hm, ESCs containing hmTert/hmTert alleles, m/hm, heterozygous ESCs with mTert/hmTert genes. MEF, wildtype mouse embryonic fibroblast cells. RNase A treated wildtype ESC extract was used as a negative control. MW, molecular weight control. IC, internal control. Underneath are relative intensities of all telomere bands in each lane, normalized to lysis buffer control (1).

Figure 4

Proliferation and chromosome stabilities of ESCs with mTert and hmTert alleles. (A) Long-term proliferation of ESCs. ESCs with mTert/mTert (m/m), mTert/hmTert (m/hm), hmTert/hmTert (hm/hm), or mTert homozygous knockout (−/−) were passaged every 2 days for 110 times (m/m, m/hm, & hm/hm) or 100 times (−/−). (B) Telomere length in ESCs. Telomere lengths (Cy3) were measured by quantitative FISH (qFISH) analysis and the data were analyzed using the Metasystems software package. Mean telomere lengths are indicated by vertical dash lines and numbers in parentheses (mean ± s.d.). (C) Telomere restriction fragment length analysis. Genomic DNAs were extracted from ESCs at various passages and digested with HinfI and RsaI, followed by pulsed-field gel electrophoresis and Southern blotting. MW, molecular weight. Genotypes and passage numbers are indicated above each lane. (D) Images of telomeres (Cy3) in ESCs. (E) Telomere and chromosome integrity in ESCs. Red, Cy3 for telomere; green, FAM for centromere; blue, DAPI for DNA. Arrows point to telomere signal-less chromosomal ends and stars indicate chromosomal end-end fusion.

Figure 5

mRNA expression and telomerase activity from mTert and hmTert loci during in vitro ESC differentiation. (A) Tert mRNA expression in differentiating EB cultures. Tert mRNA were measured by RT-qPCR and normalized to 18S rRNA. Light gray, mTert; dark gray, hmTert. (B) mTert mRNA levels in differentiated fibroblast-like cells. 3-day old EBs (m/hm) were passaged in DMEM with 10% fetal bovine serum for 2, 5, and 11 times. Passage 11 cells were also treated for 24 h with 200 ng/ml TSA. mRNAs from mTert and hmTert loci were measured using specific primers spanning intron 2. (C) Telomerase activities in ESCs and differentiated cells. Telomerase activities in extracts of 2000 or 20 ESCs and fibroblast-like cells (passage 11) were measured using TRAP assay. Numbers below are relative intensities of all telomere bands in each lane, normalized to lysis buffer control (1). (D) Covalent histone modifications at genomic regions around the hmTert and mTert promoters in ESCs and differentiated cells. Chromatin fragments from ESCs (m/hm) and differentiated cells (DIFF) were precipitated using antibodies against specific histone marks, followed by qPCR analysis. H3Ac and H4Ac refer to actylated histones H3 and H4. H3K27Ac and H3K4me3 are acetylated K27 and trimethylated K4 residues of histone H3, respectively. Pro, hmTert or mTert promoters. Up5k, Up2k, and Dn2k are chromosomal sites 5-kb and 2-kb upstream and 2-kb downstream of the Tert promoters, respectively.

Figure 6

The impact of human intron 6 on hmTert splicing. (A) PCR analysis of mTert, hmTert, and hTERT mRNAs. Total RNAs from mouse and human ESCs were subjected to PCR analysis using primers spanning introns 6–8 and analyzed on an Agarose gel. Numbers on the left are sizes of molecular weight markers in bp. (B) Diagrams of the genomic regions spanning exons 6–9. Mouse exons and introns are designated by grey lines and boxes and human sequences are shown as black lines and boxes. Splicing patterns are indicated by thin lines below. Arrowheads show the positions of PCR primers used in panel (A,C). Alternative splicing of exon 7 at the mTert and hmTert loci. mTert mRNA levels in ESCs were determined by RT-qPCR analysis using primers spanning intron 6 (FL) or introns 6–7 (∆E7). (D) Splicing of exon 7 of the hTERT locus. hTERT mRNA levels in human H9 ESCs were measured by RT-qPCR analysis using primers spanning intron 6 (FL), introns 6–7 (∆E7), or introns 6–8 (∆E7-8).