Skip to main content
. 2020 Sep 18;10:15348. doi: 10.1038/s41598-020-72216-y

Figure 2.

Figure 2

A high transgene copy number is required to promote targeted integration in adult cardiomyocytes. (A) AAV6 was directly injected into the free left ventricular wall. At 14 and 28 days after injection, heart tissues were excised and stained with anti-troponin I antibody (green). Scale bar: 300 μm. (B) The proportion of the injection area in the myocardium that was tdTomato positive, as estimated by the distribution of tdTomato fluorescent signals, were calculated using BZ-X analyzer (Keyence) (n = 3, means ± SD). *: p < 0.05, #: p < 0.01 vs day 5. (C) The area of a frozen tissue section positive for tdTomato at day 28 after injection was excised using laser micro dissection. Cross-sections before (left) and after (right) dissection are shown. Scale bar: 100 μm. A remote area was excised as control. RNA was extracted from both sections and intron-spanning PCR was performed using the cDNA samples. Original full image of the gel is presented in Supplementary Fig. 6B. (D) Tissue sections obtained at day 28 after AAV injection were immunostained and observed by confocal microscopy. Scale bar: 20 μm. (E) Genomic DNA was extracted from the dissected tissue sections and quantitative real-time PCR was performed. Forward and reverse primers were designed to amplify the ITR sequence of AAV. The numbers of transgenes were calculated as ITR copy numbers normalized by the amount of genomic DNA used as the PCR template. (F) Neonatal cultured cardiomyocytes isolated from Cas9 knock-in mice were transduced with AAV6 encoding gRNA and repair template DNA. Five days after transduction, genomic DNA were extracted. Left ventricular free wall of 8-week old Cas9 knock-in mice were injected with AAV6. Five days after injection, genomic DNA were extracted. Both experiments were performed in duplicate. The PCR-amplified genomic DNA were analyzed by deep sequencing. Indel ratio normalized by the average coverage around the PAM sequence are shown. (G) Average coverage (read/bp), ratio of NHEJ, targeted integration and proportion of targeted integration/NHEJ were analyzed in neonatal cultured cardiomyocytes and adult heart tissues.