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. 2023 Oct 21;13(1):118.
doi: 10.1186/s13568-023-01624-w.

Effects of hTERT transfection on the telomere and telomerase of Periplaneta americana cells in vitro

Chenjing Ma  1   2 , Xian Li  1 , Weifeng Ding  1 , Xin Zhang  3 , Hang Chen  1 , Ying Feng  1
Affiliations

Effects of hTERT transfection on the telomere and telomerase of Periplaneta americana cells in vitro

Chenjing Ma et al. AMB Express. .

Abstract

Telomere and telomerase are crucial factors in cell division and chromosome stability. Telomerase activity in most cells depends on the transcription control by the telomerase reverse transcriptase (TERT). The introduction of an exogenous human TERT (hTERT) in cultured cells could enhance telomerase activity and elongate the lifespan of various cells. Telomere elongation mechanisms vary between insects and are complex and unusual. Whether the use of exogenous hTERT can immortalize primary insect cells remains to be investigated. In this study, we used a recombinant virus expressing hTERT to infect primary cultured cells of Periplaneta americana and evaluated its effects on insect cell immortalization. We found that hTERT was successfully expressed and promoted the growth of P. americana cells, shortening their doubling time. This was due to the ability of hTERT to increase the activity of telomerase in P. americana cells, thus prolonging the telomeres. Our study lays the foundation for understanding the mechanisms of telomere elongation in P. americana, and suggests that the introduction of hTERT into insect cells could be an efficient way to establish certain insect cell lines.

Keywords: Cell immortalization; Periplaneta americana; Telomerase; Telomere; hTERT.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
AcMNPV-hTERT infection in RIRI-PA1-3 and RIRI-PA1-50. (A) Infection of RIRI-PA1-3 cells with AcMNPV-hTERT for 7 and 14 days (200×) at MOIs of 1, 5, and 10. (B) Infection of RIRI-PA1-50 cells with AcMNPV-hTERT for 7 and 14 days (200×) at MOIs of 1, 5, and 10. Scale bar: 100 μm
Fig. 2
Fig. 2
Changes in cell number in RIRI-PA1-3 and RIRI-PA1-50 after AcMNPV-hTERT infection for 1 to 10 days. (A) Changes in cell number in RIRI-PA1-3 cells with AcMNPV-hTERT infected for 1 to 10 days at MOIs of 1, 5, and 10. (B) Changes in cell number in RIRI-PA1-50 cells with AcMNPV-hTERT infected for 1 to 10 days at MOIs of 1, 5, and 10. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.05, and ***P < 0.001, compared with control group
Fig. 3
Fig. 3
Cell growth state of RIRI-PA1-10 and RIRI-PA-hTERT-10. (A) Control group of RIRI-PA1-10 cells (200×). (B) Infection of RIRI-PA1-10 cells with AcMNPV-hTERT MOI 10 (200×). Scale bar: 100 μm
Fig. 4
Fig. 4
Growth curve of RIRI-PA1-10 and RIRI-PA-hTERT-10. Values are expressed as mean ± SEM. *P < 0.05 and **P < 0.05, compared with control group
Fig. 5
Fig. 5
Alignment of the COI gene nucleotide sequences from P. americana embryos and RIRI-PA1 cells
Fig. 6
Fig. 6
hTERT expression in RIRI-PA1-3 and RIRI-PA1-50 after AcMNPV-hTERT infection for 7 and 14 days. (A–B) hTERT relative mRNA expression in RIRI-PA1-3 (A) and RIRI-PA1-50 (B) infected with AcMNPV-hTERT for 7 and 14 days. Values are expressed as mean ± SEM. *P < 0.05 and ***P < 0.001, compared with 7 d MOI 1 group. (C–D) Western blot analysis of recombinant hTERT protein expression in RIRI-PA1-3 and RIRI-PA1-50 infected with AcMNPV-hTERT for 7 and 14 days
Fig. 7
Fig. 7
hTERT expression in RIRI-PA1-3 and RIRI-PA1-50 leads to an increased telomerase activity 7 days after infection. (A) Telomerase activity was detected using TRAP in RIRI-PA1-3 and RIRI-PA1-50 after AcMNPV-hTERT infection for 7 days. (B) Relative telomerase activity was detected using qPCR in RIRI-PA1-3 cells 7 days after AcMNPV-hTERT infection. Values are expressed as mean ± SEM, *P < 0.05, MOI 10 group compared with control group in RIRI-PA1-3 cells. (C) Relative telomerase activity was detected using qPCR in RIRI-PA1-50 cells 7 days after AcMNPV-hTERT infection. Values are expressed as mean ± SEM, **P < 0.01, MOI 10 group compared with control group in RIRI-PA1-50 cells
Fig. 8
Fig. 8
Sequences of TRAP products obtained upon AcMNPV-hTERT infection in RIRI-PA1-3 and RIRI-PA1-50 cells
Fig. 9
Fig. 9
FISH mapping of RIRI-PA1-3 and RIRI-PA1-50 cells 7 days after AcMNPV-hTERT infection using the (TTAGG)5 telomeric probe. (A) Cy3-tagged telomeres in RIRI-PA1-3 cells 7 days after AcMNPV-hTERT infection. (B) Cy3-tagged telomeres in RIRI-PA1-50 cells 7 days after AcMNPV-hTERT infection. (C) Relative fluorescence-integrated densities of Cy3-tagged telomeres in RIRI-PA1-3. Values are expressed as mean ± SEM, ***P < 0.001, MOI 10 group compared with control group in RIRI-PA1-3. (D) Relative fluorescence-integrated densities of Cy3-tagged telomeres in RIRI-PA1-50. Values are expressed as mean ± SEM, **P < 0.01, MOI 10 group compared with control group in RIRI-PA1-50
Fig. 10
Fig. 10
Flow-chart summary of the article
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