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Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes

Hindul, Naushin L., Jhita, Amarjot, Oprea, Daiana G., Hussain, Tasnim Alamgir, Gonchar, Oksana, Campillo, Miguel Angel Muro, O'Regan, Laura, Kanemaki, Masato T., Fry, Andrew M., Hirota, Kouji, Tanaka, Kayoko·2022·Biology Open
DOI 10.1242/bio.059056PMC PMC8864296
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Abstract

The human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes.

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Figures

Fig. 1. Construction of hTERT RPE-1 ERT2-Cre-ERT2 cell line. (A) A schematic diagram of the integration donor plasmid pInt-ERCreER and the targeted AAVS1 locus. Locations of the primers used to genotype the integration event are indicated in blue. (B) Representative genome PCR results using primers Pur-F and AAVS1arm2-chk-R. Successful integration of the ERT2-Cre-ERT2 cassette into AAVS1 locus of at least one of the chromosomes yields a 1330 bp PCR product (indicated by a blue arrowhead). Numbers indicated for each lane represent clones isolated through the puromycin resistance screening. Clones 19 and 65 (shown in green) were further analysed in this study. BG indicates background bands. (C) Representative genome PCR results using primers AAVS1arm1-chk-F and AAVS1arm2-chk-R. This pair of primers amplifies an 1836 bp fragment (indicated by a blue arrowhead) if the wild-type unedited AAVS1 locus is present. The result indicates that clones 19, 20, 48, 51, 53, 54 and 65 do not retain the wild-type AAVS1 locus. Together with the result from panel B, we concluded that these clones carry the ERT2-Cre-ERT2 cassette as a homozygous insertion at the AAVS1 locus. Clones 19 and 65 were further analysed for Cre expression and activity in Figs 2 and 3. BG indicates background bands.
Fig. 1. Construction of hTERT RPE-1 ERT2-Cre-ERT2 cell line. (A) A schematic diagram of the integration donor plasmid pInt-ERCreER and the targeted AAVS1 locus. Locations of the primers used to genotype the integration event are indicated in blue. (B) Representative genome PCR results using primers Pur-F and AAVS1arm2-chk-R. Successful integration of the ERT2-Cre-ERT2 cassette into AAVS1 locus of at least one of the chromosomes yields a 1330 bp PCR product (indicated by a blue arrowhead). Numbers indicated for each lane represent clones isolated through the puromycin resistance screening. Clones 19 and 65 (shown in green) were further analysed in this study. BG indicates background bands. (C) Representative genome PCR results using primers AAVS1arm1-chk-F and AAVS1arm2-chk-R. This pair of primers amplifies an 1836 bp fragment (indicated by a blue arrowhead) if the wild-type unedited AAVS1 locus is present. The result indicates that clones 19, 20, 48, 51, 53, 54 and 65 do not retain the wild-type AAVS1 locus. Together with the result from panel B, we concluded that these clones carry the ERT2-Cre-ERT2 cassette as a homozygous insertion at the AAVS1 locus. Clones 19 and 65 were further analysed for Cre expression and activity in Figs 2 and 3. BG indicates background bands.CC BY · PubMed Central
Fig. 2. Expression of ERT2-Cre-ERT2 is induced within 10 h after doxycycline treatment. hTERT RPE-1 ERT2-Cre-ERT2 clones 19 and 65 were treated with doxycycline (1 µg/ml) and the expression levels of ERT2-Cre-ERT2 at time 0, 10, 24 and 48 h after the treatment were analysed by western blotting. (A) A representative result of the parental hTERT RPE-1, clone 19 and clone 65. (B) Three biological replicates of the western blotting were quantitated using actin as an internal control. Mean and SD values are presented.
Fig. 2. Expression of ERT2-Cre-ERT2 is induced within 10 h after doxycycline treatment. hTERT RPE-1 ERT2-Cre-ERT2 clones 19 and 65 were treated with doxycycline (1 µg/ml) and the expression levels of ERT2-Cre-ERT2 at time 0, 10, 24 and 48 h after the treatment were analysed by western blotting. (A) A representative result of the parental hTERT RPE-1, clone 19 and clone 65. (B) Three biological replicates of the western blotting were quantitated using actin as an internal control. Mean and SD values are presented.CC BY · PubMed Central
Fig. 3. Cre-LoxP excision of the targeted chromosome in 48 h after doxycycline and 4-OHT treatment. (A) A schematic diagram of a proof-of-principle test case, chromosome editing of the KRAS gene locus. A DNA fragment encoding histidinol dehydrogenase (HDH), flanked by LoxP sites (LoxP-HDH-LoxP, LHL) was inserted into intron 2 of KRAS gene to create KRAS.LHL locus. Active Cre is expected to excise the LHL cassette, leaving one copy of LoxP site, to generate KRAS.LoxP allele. A pair of primers, AlugenomecheckF and CheckR, is indicated in the diagram. Two probes used for the Southern blotting are indicated as ‘SB probes’ and EcoRI sites used for Southern blotting are indicated in the diagram. (B) Some of the clones with the LoxP cassette insertion also carried the KRAS.G12V mutation. The genome isolated from clone 19-16, which has a heterozygous integration of the ERT2-Cre-ERT2 cassette, was sequenced using the primer AlugenomecheckF. Three substitutions of the nucleic acids resulting in G12V mutation are indicated with orange arrows together with the sequencing chromatogram. (C) A typical example of successful excision of the LHL cassette 48 h after doxycycline (1 µg/ml) and 4-OHT (0.5 µM) exposure in seven independent clones. Clones 19-16, 19-12, 19-13 and 19-21 were generated by integrating the LHL cassette into the KRAS locus of clone 19 and clones 65-16, 65-30 and 65-32 were generated in the same way but using clone 65. Among these clones, clones 19-21 and 65-30 have KRAS.LHL as a homozygous insertion. PCR genotyping was conducted using primers AlugenomecheckF and CheckR. The PCR reaction produced the expected products of 4621 bp for KRAS.LHL, 2370 bp for KRAS.wt and 2115 bp for KRAS.LoxP. (D) Southern blotting to confirm the successful LHL excision 48 h after doxycycline and 4-OHT treatment. Genomes from clone 19 and clone 19-16 were digested by EcoRI and analysed by Southern blotting using a cocktail of two probes indicated in the diagram A (SB probes). Signals were detected at the expected size of 8797 bp for KRAS.LHL. For KRAS.wt and KRAS.LoxP, the expected sizes were 6546 bp and 6291 bp, respectively, and these two bands were not resolved in the agarose gel. Nonetheless, the KRAS.LHL signal became undetectable in clone 19-16 (time 48) sample, and therefore we concluded that LHL excision was successful 48 h after exposure to doxycycline and 4-OHT. (E) Time-course profile of the LHL excision. Parental hTERT RPE-1, clone 19 and clone 19-16 were exposed to doxycycline (1 µg/ml) and 4-OHT (0.5 µM), and samples collected at 0, 10, 24 and 48 h after treatment. Genome PCR was conducted using primers AlugenomecheckF and CheckR. A PCR product of KRAS.LoxP was detectable 10 h after the start of the treatment. By 48 h, most of the KRAS.LHL signal disappeared and the signal intensity of KRAS.wt and KRAS.LoxP became comparable.
Fig. 3. Cre-LoxP excision of the targeted chromosome in 48 h after doxycycline and 4-OHT treatment. (A) A schematic diagram of a proof-of-principle test case, chromosome editing of the KRAS gene locus. A DNA fragment encoding histidinol dehydrogenase (HDH), flanked by LoxP sites (LoxP-HDH-LoxP, LHL) was inserted into intron 2 of KRAS gene to create KRAS.LHL locus. Active Cre is expected to excise the LHL cassette, leaving one copy of LoxP site, to generate KRAS.LoxP allele. A pair of primers, AlugenomecheckF and CheckR, is indicated in the diagram. Two probes used for the Southern blotting are indicated as ‘SB probes’ and EcoRI sites used for Southern blotting are indicated in the diagram. (B) Some of the clones with the LoxP cassette insertion also carried the KRAS.G12V mutation. The genome isolated from clone 19-16, which has a heterozygous integration of the ERT2-Cre-ERT2 cassette, was sequenced using the primer AlugenomecheckF. Three substitutions of the nucleic acids resulting in G12V mutation are indicated with orange arrows together with the sequencing chromatogram. (C) A typical example of successful excision of the LHL cassette 48 h after doxycycline (1 µg/ml) and 4-OHT (0.5 µM) exposure in seven independent clones. Clones 19-16, 19-12, 19-13 and 19-21 were generated by integrating the LHL cassette into the KRAS locus of clone 19 and clones 65-16, 65-30 and 65-32 were generated in the same way but using clone 65. Among these clones, clones 19-21 and 65-30 have KRAS.LHL as a homozygous insertion. PCR genotyping was conducted using primers AlugenomecheckF and CheckR. The PCR reaction produced the expected products of 4621 bp for KRAS.LHL, 2370 bp for KRAS.wt and 2115 bp for KRAS.LoxP. (D) Southern blotting to confirm the successful LHL excision 48 h after doxycycline and 4-OHT treatment. Genomes from clone 19 and clone 19-16 were digested by EcoRI and analysed by Southern blotting using a cocktail of two probes indicated in the diagram A (SB probes). Signals were detected at the expected size of 8797 bp for KRAS.LHL. For KRAS.wt and KRAS.LoxP, the expected sizes were 6546 bp and 6291 bp, respectively, and these two bands were not resolved in the agarose gel. Nonetheless, the KRAS.LHL signal became undetectable in clone 19-16 (time 48) sample, and therefore we concluded that LHL excision was successful 48 h after exposure to doxycycline and 4-OHT. (E) Time-course profile of the LHL excision. Parental hTERT RPE-1, clone 19 and clone 19-16 were exposed to doxycycline (1 µg/ml) and 4-OHT (0.5 µM), and samples collected at 0, 10, 24 and 48 h after treatment. Genome PCR was conducted using primers AlugenomecheckF and CheckR. A PCR product of KRAS.LoxP was detectable 10 h after the start of the treatment. By 48 h, most of the KRAS.LHL signal disappeared and the signal intensity of KRAS.wt and KRAS.LoxP became comparable.CC BY · PubMed Central
Fig. 4. The physiological impact of expression of ERT2-Cre-ERT2 in the clone 19. (A–C) Proliferation rates of RPE1 and clone 19 cells with or without doxycycline and 4-OHT treatment. A relatively small number of cells (2×103 cells) were seeded per well in a six-well plate to allow observation of proliferation for up to 8 days. The panels show the proliferation rates from day 5. The cell count data for the whole course of the experiment is presented in the Fig. S2. All graphs have a logarithmic y-axis. (A) Proliferation rates of RPE1 and clone 19 cells without doxycycline and 4-OHT treatment. (B) Proliferation rates of RPE1 and clone 19 cells cultured in the presence of doxycycline and 4-OHT throughout the experiment. (C) Proliferation rates of RPE1 and clone 19 cells treated for 48 h with doxycycline and 4-OHT before replacement into fresh media. For the clone 19 samples treated with doxycycline and 4-OHT, cell counts were made for an additional day to reveal growth recovery of these samples. All experiments, n=3; error bars represent SD. (D) Formation of γ-H2AX foci upon expression of ERT2-Cre-ERT2. RPE-1 and Clone 19 cells were subject to the following treatments: incubation for 48 h with doxycycline and 4-OHT, incubation for 48 h with doxycycline and 4-OHT, which were then removed for 24 h before fixation, and γ-irradiation (0.5 Gy) 30 min before fixation. Untreated cells are included as a reference. γ-H2AX foci formation was detected by immunofluorescence microscopy as described in the Materials and Methods. Upper panels show merged images of γ-H2AX foci and DNA. Scale bar: 20 µm. (E) Summary of γ-H2AX foci formation results presented in (D). 100–150 nuclei were counted for each sample and the data presented using the boxplot format (RStudio) where median, and first and third quartiles are indicated using a box. Individual data points are also indicated. The data were analysed by Kruskal–Wallis test and pairwise comparisons conducted by Dunn’s test. NS, not significant; **P<0.01, ***P<0.001, ****P<0.0001, *****P<0.00001.
Fig. 4. The physiological impact of expression of ERT2-Cre-ERT2 in the clone 19. (A–C) Proliferation rates of RPE1 and clone 19 cells with or without doxycycline and 4-OHT treatment. A relatively small number of cells (2×103 cells) were seeded per well in a six-well plate to allow observation of proliferation for up to 8 days. The panels show the proliferation rates from day 5. The cell count data for the whole course of the experiment is presented in the Fig. S2. All graphs have a logarithmic y-axis. (A) Proliferation rates of RPE1 and clone 19 cells without doxycycline and 4-OHT treatment. (B) Proliferation rates of RPE1 and clone 19 cells cultured in the presence of doxycycline and 4-OHT throughout the experiment. (C) Proliferation rates of RPE1 and clone 19 cells treated for 48 h with doxycycline and 4-OHT before replacement into fresh media. For the clone 19 samples treated with doxycycline and 4-OHT, cell counts were made for an additional day to reveal growth recovery of these samples. All experiments, n=3; error bars represent SD. (D) Formation of γ-H2AX foci upon expression of ERT2-Cre-ERT2. RPE-1 and Clone 19 cells were subject to the following treatments: incubation for 48 h with doxycycline and 4-OHT, incubation for 48 h with doxycycline and 4-OHT, which were then removed for 24 h before fixation, and γ-irradiation (0.5 Gy) 30 min before fixation. Untreated cells are included as a reference. γ-H2AX foci formation was detected by immunofluorescence microscopy as described in the Materials and Methods. Upper panels show merged images of γ-H2AX foci and DNA. Scale bar: 20 µm. (E) Summary of γ-H2AX foci formation results presented in (D). 100–150 nuclei were counted for each sample and the data presented using the boxplot format (RStudio) where median, and first and third quartiles are indicated using a box. Individual data points are also indicated. The data were analysed by Kruskal–Wallis test and pairwise comparisons conducted by Dunn’s test. NS, not significant; **P<0.01, ***P<0.001, ****P<0.0001, *****P<0.00001.CC BY · PubMed Central

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Citations

Cites

Research works printed but not linked: 32

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Metadata

Authors
Hindul, N. L., Jhita, A., Oprea, D. G., Hussain, T. A., Gonchar, O., Campillo, M. A. M., O'Regan, L., Kanemaki, M. T., Fry, A. M., Hirota, K., & Tanaka, K.
Venue
Biology Open
Publisher
The Company of Biologists
Published
2022-02-15
Volume
11
Issue
2
ISSN
2046-6390
DOI
10.1242/bio.059056
PMC
PMC8864296
Cited by
12
References
3
Language
en
Licence
creativecommons.org/licenses/by/4.0 (vor, 1 d)
Funders
  • JSPS (JP20H04337, JP16H01314, JP19KK0210)
  • University of Leicester

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