Dissecting the roles of miR-302/367 cluster in cellular reprogramming using TALE-based repressor and TALEN.

MicroRNAs are important gene regulators involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs mainly include locked nucleic acid (LNA) oligonucleotides and miRZip inhibitors, which have several limitations. Due to their unique gene structures and small sizes, there is no efficient or simple strategy to knock down or knock out microRNAs or whole microRNA clusters. Here, we demonstrate knockdown of the miR-302/367 cluster by using the Kruppel-associated box repressor domain fused with specific transcription activator-like effectors (TALEs) designed to bind the miR-302/367 cluster promoter. We also designed two pairs of TALE nucleases (TALENs) to efficiently delete the miR-302/367 cluster in primary human fibroblasts and determined that knockout of the miR-302/367 cluster completely blocked induced pluripotent stem cell (iPSC) generation. Together, our results demonstrate that TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA clusters in somatic cells and pluripotent stem cells.

Coexpression of angiopoietin-1 with VEGF increases the structural integrity of the blood-brain barrier and reduces atrophy volume.

Vascular endothelial growth factor (VEGF)-induced neovasculature is immature and leaky. We tested if coexpression of angiopoietin-1 (ANG1) with VEGF improves blood-brain barrier (BBB) integrity and VEGF neuroprotective and neurorestorative effects using a permanent distal middle cerebral artery occlusion (pMCAO) model. Adult CD-1 mice were injected with 2 × 10(9) virus genomes of adeno-associated viral vectors expressing VEGF (AAV-VEGF) or ANG1 (AAV-ANG1) individually or together in a 1:1 ratio into the ischemic penumbra 1 hour after pMCAO. AAV-LacZ was used as vector control. Samples were collected 3 weeks later. Compared with AAV-LacZ, coinjection of AAV-VEGF and AAV-ANG1 reduced atrophy volume (46%, P=0.004); injection of AAV-VEGF or AAV-ANG1 individually reduced atrophy volume slightly (36%, P=0.08 and 33%, P=0.09, respectively). Overexpression of VEGF reduced tight junction protein expression and increased Evans blue extravasation. Compared with VEGF expression alone, coexpression of ANG1 with VEGF resulted in upregulation of tight junction protein expression and reduction of Evans blue leakage (AAV-ANG1/AAV-VEGF: 1.4 ± 0.3 versus AAV-VEGF: 2.8 ± 0.7, P=0.001). Coinjection of AAV-VEGF and AAV-ANG1 induced a similar degree of angiogenesis as injection of AAV-VEGF alone (P=0.85). Thus, coexpression of ANG1 with VEGF improved BBB integrity and resulted in better neuroprotection compared with VEGF expression alone.