Cfx96 system

The DNA extracted from the cervix of the pigs before the admistration and on the seventh day after admistration was used to conduct the Real-time PCR, and the qPCR was conducted using the Bio-Rad CFX96 system with PowerUp™ SYBR™ Green Master Mix (A25742, Applied Biosystems, USA). The experiments were performed in triplicates, and the relative expression change of PERV copy number was calculated using the comparative Ct method (which compared the Ct value before and after admistration) with GAPDH of pig as the reference gene. The primers used were listed (Table 2).

Total RNA was extracted from Huh-7 and HA22T cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed into cDNA using Superscript III transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) at 42°C for 60 min and 95°C for 5 min. qPCR was conducted using a Bio-Rad CFX96 system (Applied Biosystems) with SYBR-Green to detect the mRNA expression level of a gene of interest using the following thermocycling conditions: 95°C for 3 min, followed by 35 cycles at 95°C for 15 sec, 60°C for 30 sec and 68°C for 1 min. Expression levels were normalized to the GAPDH levels using the 2^−ΔΔCq method (34 (link)). The primer sequences were as follows: miR-191-5p forward, 5′-CAACGGAATCCCAAAAGCAGCTG-3′ and reverse, 5′-TGTCGTGGAGTCGGC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; IGF2BP3 forward, 5′-ACTGCACGGGAAACCCATAG-3′ and reverse, 5′-ACTATCCAGCACCTCCCACT-3′; ZO-1 forward, 5′-GTGGGTAACGCCATCCTCTG-3′ and reverse, 5′-TCCGGGATTTCACCAGTGTG-3′; and GAPDH forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′. All primers were purchased from Integrated DNA Technologies, Inc.

The qPCR was performed using the Bio-Rad CFX96 system with PowerUp™ SYBR™ Green Master Mix (A25742, Applied Biosystems, USA). Threshold-cycle value (Ct value) was used for analysis for each qPCR reaction. The primers involved were displayed in Table S3.