MicroRNA-132 (miR-132) has been demonstrated to affect multiple neuronal functions, including dendritic growth and spinogenesis in cultured neurons and brain slices, as well as learning behavior of animals. However, its role in acquisition of temporal-associated memory remains unclear. In this study, we demonstrated that the mature miR-132 level in mouse hippocampus was significantly increased at 30 min after trace fear conditioning, a type of temporal-associated learning, and returned to baseline values in 2 h. We then knocked down miR-132 expression in vivo by infusing a lentivector expressing anti-miR-132 hairpin RNA into the third ventricle near the anterior hippocampi such RNA diffused laterally to both hippocampal formations, later confirmed by histological analysis. This approach successfully reduced hippocampal miR-132 expression in both naïve and trace fear conditioned groups, and impaired acquisition of trace fear memory in mice. To our knowledge, this result is the first demonstration of change in temporal learning behavior by reducing microRNA (miRNA) level specifically in the hippocampal region.

Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

This study reports the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction‐mediated, site‐specific integration of OKSM transcription factor genes into the AAVS1 locus in human chromosome 19. Methods based on site‐specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable induced pluripotent stem cells suitable for future gene therapy applications.