Analyzing differentially expressed genes and pathways of Bex2-deficient mouse lung via RNA-Seq.

Bex2 is well known for its role in the nervous system, and is associated with neurological disorders, but its role in the lung's physiology is still not reported. To elucidate the functional role of Bex2 in the lung, we generated a Bex2 knock-out (KO) mouse model using the CRISPR-Cas9 technology and performed transcriptomic analysis. A total of 652 genes were identified as differentially expressed between Bex2 -/- and Bex2 +/+ mice, out of which 500 were downregulated, while 152 were upregulated genes. Among these DEGs, Ucp1, Myh6, Coxa7a1, Myl3, Ryr2, RNaset2b, Npy, Enob1, Krt5, Myl2, Hba-a2, and Nrob2 are the most prominent genes. Myl2, was the most downregulated gene, followed by Npy, Hba-a2, Rnaset2b, nr0b2, Klra8, and Ucp1. Tcte3, Eno1b, Zfp990, and Pcdha9 were the most upregulated DEGs. According to gene enrichment analysis, PPAR pathway, cardiac muscle contraction, and cytokine-cytokine receptor interaction were the most enriched pathways. Besides, the nuclear factor-κB signaling pathway and hematopoietic cell linage pathways were also enriched. Chronic obstructive pulmonary disease (COPD) is enriched among KEGG disease pathways. RT-qPCR assays confirmed the RNA-Seq results. This study opens a new window toward the biological functions of Bex2 in different systems.

Effect of low VEGF on lung development and function.

Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship between VEGF expression levels and its proper development and pathogenesis. Human SPC promoter has been used to faithfully express genes or cDNAs in the pulmonary epithelium in many transgenic mouse models. In the study, a mouse model of lung-specific and reversible VEGF repression (hspc-rtTRtg/+/VegftetO/tetO) was generated. Human SPC promoter was used to drive lung-specific rtTR expression, a cDNA coding for doxycycline-regulated transcription repression protein. By crossing with VegftetO/tetO mice, that has tetracycline operator sequences insertion in 5'-UTR region, it allows us to reversibly inhibit lung VEGF transcription from its endogenous level through doxycycline food, water or injection. The tissue-specific inhibition of VEGF is used to mimic abnormal expression levels of VEGF in lung. Reduced VEGF expression in lung is confirmed by quantitative real time PCR and immunoblotting. Lung development and structure was analyzed by histology analysis and found significantly affected under low VEGF. The pulmonary epithelium and alveolarization are found abnormal with swelling alveolar septum and enlargement of air space. Genome-wide gene expression analysis identified that immune activities are involved in the VEGF-regulated lung functions. The transgenic mouse model can be used to mimic human pulmonary diseases. The mouse model confirms the important regulatory roles of epithelial expressed VEGF in lung development and function. This mouse model is valuable for studying VEGF-regulated lung development, pathogenesis and drug screening under low VEGF expression.

Brain transcriptome study through CRISPR/Cas9 mediated mouse Dip2c gene knock-out.

Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including 'memory', 'neuropeptide signaling pathway', and 'response to amphetamine' while KEGG analysis shows that 'neuroactive ligand-receptor interaction pathway' is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.