Animals

Three- to four-month-old outbred OF1 mice (maintained in Institute of Zoology and Biomedical Research, Jagiellonian University, Kraków, Poland) were used for the experiments. The experiments were conducted at the Institute of Zoology and Biomedical Research of the Jagiellonian University according to the guidelines of the European Community Directive 86/609 and to the Polish Governmental Act on Animal Protection. The animals were maintained in a temperature- and light-controlled room (22 °C, 12-h light–dark cycle) and were provided with food and water ad libitum.

Cells

Mouse fibroblasts (NIH/3T3 cell line; ATCC No. CRL-1658) in a concentration of 106/ml, grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Paisley, Scotland), penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (Sigma Aldrich), were obtained from Department of Cell Biology; Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Kraków, Poland.

DNA extraction from tail tissue and its fragmentation

DNA from the tip of a mouse tail was isolated with the GeneMATRIX Tissue DNA Purification Kit (EURx, Gdańsk, Poland) in accordance with the manufacturer’s instruction. The purity of the isolate (260/280 and 260/230 ratios) was verified on a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then divided into four equal parts. One part remained untreated (control DNA), and the three others were sheared in a Bioruptor Pico (Diagenode, Denville, NJ, USA) using 1, 2, or 3 sonication cycles (5/30 s on/off). The integrity of the control DNA and fragmentation of the sonicated DNA were confirmed by agarose gel electrophoresis (Figure S1). The fragmented DNA samples were measured on the Qubit Fluorometer (Invitrogen, Waltham, MA, USA) using the Qubit 1X dsDNA HS Assay Kit (Invitrogen), then diluted to a concentration of 1 ng/μl, and analyzed with a 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA) to determine the average length of the generated fragments. The same samples (diluted to a concentration of 0.5 ng/μl) were reanalyzed with the LORD-Q assay using the same procedure as for single cells.

Collection of oocytes

Oocyte isolation and further handling were performed under stereomicroscope using mouth pipette as previously described (e.g. Polanski et al. 2005). Female mice were killed by cervical dislocation, and their ovaries were dissected and placed in M2 culture medium supplemented with 150 μg/ml dbcAMP (Sigma-Aldrich) to prevent meiosis resumption. The oocytes were then released from the ovaries by puncturing the follicles and collected in a drop of M2 medium with dbcAMP under mineral oil. Only oocytes at the first meiotic prophase stage (with visible germinal vesicle, GV) were used for further procedures.

Induction of DNA damage in oocytes and fibroblasts

GV oocytes in a 100 μl M2 drop or DMEM suspension of fibroblasts diluted 10X with M2 medium (100 μl) were exposed to ultraviolet C (UV-C) radiation (wavelength 254 nm) at a dose of 15, 30, 60, or 120 mJ/cm2 in a UV crosslinker (UVP HL-2000 HybriLinker, UVP, Upland, CA, USA). After irradiation, single fibroblasts were individually collected in the buffer for DNA extraction, while single oocytes were collected either for DNA extraction or for immunofluorescent staining.

Immunofluorescent staining of oocytes and confocal microscopy

The oocytes were briefly washed in PBS and then fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. After washing in PBS (3× for 10 min), the oocytes were permeabilized for 15 min in 0.1% Triton X 100 in PBS and incubated in 10% fetal bovine serum (FBS) at room temperature. Then, the oocytes were incubated overnight at 4 °C with the primary anti-γ-H2AX antibody (Cell Signaling Technology, Danvers, MA, USA, 9718) diluted 1 : 100 in 10% FBS. After washing in PBS (3× for 10 min), the oocytes were incubated for 1 h at room temperature with secondary Cy3-conjugated goat anti-rabbit antibody (Abcam, Cambridge, UK, ab6939), used at a dilution of 1 : 500 in 10% FBS. For nuclear DNA visualization, after washing in PBS (3× for 10 min), the oocytes were incubated for 15 min in HCS NuclearMask™ (Thermo Fisher Scientific), then washed in PBS for 10 minutes, and mounted in 1 μl PBS droplets using a Vaseline layer between a slide and a cover glass to preserve the oocyte three-dimensional structure. The mounted specimens were analyzed with the ZEISS LSM 880 Confocal Laser Scanning Microscope (Oberkochen, Germany) using the 20× Zeiss Plan-Apochromat Infinitive corrected objective with a numerical aperture of 0.8. Fluorescence signal intensities from irradiated oocytes were measured using ImageJ software and normalized to control (non-irradiated oocytes).

DNA extraction from oocytes and fibroblasts

Single control or irradiated oocytes/fibroblasts were placed individually, with the help of mouth pipette, in 0.2 ml tubes containing 40 μl of Taq Buffer with KCl (Thermo Fisher Scientific) and proteinase K (200 μg/ml) (Sigma-Aldrich). Cells in the lysis buffer were heated at 55 °C for 30 min followed by heating at 95 °C for 10 min for proteinase K deactivation. The lysates were then stored at −20 °C until the real-time PCR analysis.

Preparation of the standard of L1 copy number

Long fragment (3494 bp) of L1 was amplified with PCR using Takara LA Taq DNA Polymerase Hot-Start Version (Shiga, Japan) and primers designed for the L1-LORD-Q assay at a concentration of 200 nM each. The reaction was carried out in a volume of 50 μl on 250 ng of input DNA isolated from mouse tail tissue. The cycling conditions were as follows: (1) 1 min at 94 °C; (2) 35 cycles of 10 s at 98 °C, 30 s at 60 °C, and 3 min at 68 °C; (3) 10 min at 68 °C. The PCR product was separated from other components of the reaction by agarose gel electrophoresis and then extracted from agarose using the Gel-Out kit (A&A, Gdańsk, Poland). The purified fragment of L1 was inserted into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and cloned in JM106 E. coli cells according to the manufacturer’s instructions. Vectors were extracted from bacteria culture with the Plasmid Mini kit (A&A), and their concentration was measured in Nanodrop. The obtained plasmid was verified to have the L1 sequence by PCR, restriction enzyme digestion, and electrophoresis. After successful verification, the plasmid was diluted to a concentration of 106 copies per 40 μl and used as a standard in real-time PCR assays.

L1-LORD-Q assay

Primers designed for single-cell real-time PCRs amplify long (3494 bp) and internally nested short fragment (66 bp) of a conserved region of the L1 ORF2 sequence. The reverse primer is the same for both reactions, while the forward primers determine the length of the PCR product (Table 1). The primers were designed based on L1Base 2 (http://l1base.charite.de) containing sequences of putatively active L1 insertions residing in human and rodent genomes (Penzkofer et al. 2017). Primer specificity was confirmed by agarose gel electrophoresis and melt curve analysis (Figure S2). Taq polymerase and standard SYBR Green I dye were used for the amplification and real-time detection of the short L1 sequence. In order to ensure proper amplification of the long sequence, high-fidelity and rapid PrimeSTAR GXL polymerase was employed in combination with ResoLight dye, which is less inhibitory to DNA polymerase during long-run PCR.

Table 1 Primers used for the L1-LORD-Q assay and mean PCR efficiencies (± SD)

The reaction mixture for the short fragment included 2 μl of DNA template, PowerUp SybrGreen Master Mix (Applied Biosystems, USA), and 200 nM of each primer in a total volume of 10 μl per well. Real-time analysis was performed on the QuantStudio 5 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The cycling conditions were as follows: (1) 2 min at 50 °C, (2) 2 min at 95 °C, (3) 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Next, a melt curve was drawn to ensure that there was no unspecific amplification and/or primer–dimer formation. The reaction mixture for the long fragment included 2 μl of DNA template, 0.4 μl of PrimeSTAR GXL Polymerase (Takara), PrimeSTAR GXL Buffer (Takara), dNTP mix (200 μM each) (Takara), primers (200 nM each), and 0.05 of ResoLight Dye (Roche, Basel, Switzerland) in a total volume of 10 μl per well. Real-time analysis was carried out on the LightCycler 96 System (Roche). The cycling conditions were as follows: (1) 1 min at 98 °C, (2) 40 cycles of 10 s at 98 °C, 15 s at 60 °C, and 1 35 s at 68 °C. Next, a melt curve was drawn to ensure that there was no unspecific amplification and/or primer–dimer formation.

In each 96-well PCR plate, apart from samples, serial two-fold dilutions of the copy number standard were amplified (7 points from 5 × 105 to 7.8 × 103copies). On the basis of standard curves, the efficiency of the reactions in the subsequent plates was calculated (Figure S3). The standard curves created for the amplification of the short fragment were additionally used to assess the number of L1 sequences amplified in a single-cell genome during the assay. All DNA samples were run in triplicate. Mean Ct values derived from real-time PCR runs together with amplification efficiencies were used to calculate the number of DNA lesions per 10 kb in the examined material. For the above calculations, the equation introduced by Lehle et al. (2014) was used:

$$\frac}}=10\ 000\times \left[^\left(\textrm\right)}\times ^\left(\textrm\right)}}\left(^\left(\textrm\ 1\right)}\times ^\left(\textrm\ 1\right)}\right)\times \\ \dots \times \left(^\left(\textrm\ n\right)}\times ^\left(\textrm\ n\right)}\right)\end\right]}^}\right)}^-1\right]$$

EL:

amplification efficiency of the long fragment

ES:

amplification efficiency of the short fragment

CtL:

threshold cycle of the long fragment

CtS:

threshold cycle of the short fragment

a:

number of base pairs of the long fragment

n:

number of control samples

Statistics

All sets of data were checked for Gaussian distribution with the Shapiro–Wilk test. Since not all data appeared to be normally distributed, differences between groups were analyzed with the nonparametric Kruskal–Wallis test. P value below 0.05 was considered statistically significant.