- Alvarez et al., 2005). - All BEX genes consist of three exons, but only the third exon is coding for the protein (Fernandez et al., 2015).. - hBEX2 gene was identified in 2002 in the embryonic cerebral cDNA library using high throughput sequencing technique (Yang et al., 2002), and mBex2 in 1999 (Brown and Kay, 1999). - A robust expression of Bex2 has been observed in the mouse embryonic brain that plays a vital role in the development of the nervous system. - BEX2 protein interacts with the hematopoietic transcription factor LIM domain only 2 (LMO2) and regulates transcriptional activity of an E-box sequence-binding complex that contains hBex2, LMO2, NSCL2 and LDB1 (Han et al., 2005). - differentially expressed in breast tumors (Naderi et al., 2007). - BEX2 activates the NF-kB pathway and thus inhibits ceramide 2 (C2) apoptosis of breast cancer cells (Naderi et al., 2007). - An other group of researchers have identified that silencing of this gene promotes colorectal cancer metastasis through the Hedgehog signaling pathway (Tan et al., 2020).. - Bex2 is detected to be an oncogene in multiple types of cancers (Kazi et al., 2015). - A genome-wide association study (GWAS) revealed that Bex2 might act as a tumor inhibitor in glioma’s virulence (Foltz et al., 2006).. - propagation via NF-kB signaling pathway (Meng et al., 2014). - A very recent study explains the involvement of Bex2 in the amelioration of allergic airway inflammation by adipose stem cell derived vesicles (Kim et al., 2020).. - Abstract: 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. - Gene expression analysis has found that Bex2 is highly enriched in the lungs, but its function in the lungs has never been studied.. - The transcriptomic analysis utilizes the second- generation DNA sequencing technology to measure the genome-wide gene expression levels (Costa et al., 2010). - RNA-seq has been extensively used as a comprehensive approach to investigate various cell types and transcriptional patterns in transgenic mouse models (Adlat et al., 2020. - Sah, et al., 2020). - It is helpful to disclose novel genes and alternative splicing events (Trapnell, et al., 2012). - Gene enrichment analysis can give a broad picture of genes involvement in pathways that predict its function and regulatory network (Kumar et al., 2016).. - Although a Bex2 knocked-out mouse has already been generated by other researchers (Ito et al., 2014), the transcriptomic analysis of lungs has not been studied to the best of our knowledge. - The care has been taken to minimize the discomfort of the experimental animals.. - The mice were kept at the 12/12 light-dark cycle rotation in the pathogen- free environment with free access to food and water. - The plasmid used, pX330-U6-Chimeric_BB-CBh- hSpCas9, was gifted from Feng Zhang (Cong et al., 2013), through Addgene (https://www.addgene.org/42230. - benchling.com), authenticated using BLAST tool of the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and annealed according to the previously described methods (Ran et al., 2013). - coli strain DH5a was used for the transformation of the ligation product. - The mixes of the two plasmids (5 ng/uL each) were microinjected to the pronucleus of the fertilized oocytes, following the previously described methods (Wang et al., 2013).. - RNA-Seq data analysis. - Low value reads, adaptor-only, and reads with more than one undisclosed base were distant using SOAPnuke (Cock et al., 2010) to acquire clean reads. - Reads were compiled into longer transcripts and aligned to reference genome (GCF GRCm38.p6) using HISAT (Kim et al., 2015) and Bowtie2 tool (Langmead and Salzberg, 2012). - Following the normalization of the data, differentially expressed genes (DEGs) were analysed with |log2FC. - To knock out the gene, the sgRNA1 was designed in front of the start codon, while the sgRNA2 (Table) was targeted downstream of the start codon but before stop codon (Figure 1A). - We performed quantitative real-time PCR (qPCR) to identify the distinctively expressed genes in the lung and brain between wild-type and knock-out mice. - The relative mRNA expression results showed loss of the Bex2 (Figure 1D).. - BGISEQ platform was used for RNA-seq. - In the first category, FPKM <=1, 4605 genes were expressed in Bex2. - In the category FPKM >=10, 6358 genes were found in Bex2. - A heatmap of the gene expression is provided (Figure 2A). - We identified a total of 652 unigenes that were differentially expressed (DEG) (152 upregulated and 500 downregulated) in the lungs between Bex2. - (CC) (Ashburner et al., 2000). - Significant DEGs in the BP category contains regulation of the signaling receptor activity, inflammatory response and oxygen transport (Figure 3A). - In the CC category, cardiac troponin complex, contractile fiber muscle myosin complex were the leading terms (Figure 3C).. - (A) Graphical representation of the Bex2 gene and targeting sites. - (B) Genotyping of the pups by PCR. - (C) Sanger sequencing chromatogram of the deletion site. - An outline of the DEGs between Bex2. - (A) Heatmap presentation of the DEGs. - (B) Statistics of the upregulated and downregulated genes. - sarcomere organization cardiac muscle contraction cardiac myofibril assembly muscle contraction cardiac muscle tissue morphogenesis skeletal muscle thin filament assembly cardiac muscle fiber development regulation of muscle contraction brown fat cell differentiation neutrophil chemotaxis heart development regulation of the force of heart contraction regulation of heart rate DNA integration ventricular cardiac muscle tissue morphogenesis. - BEX2 expression was favorably found in embryonic brain, which has a vital role in the nervous system development Figure 5. - and related neurological disorders (Han et al., 2005). - The gene is predominantly expressed in the brain, but the significant expression is found in other tissues, such as the embryonic liver, adult placenta, and lungs. - Although the Bex2 KO mouse model has been generated (Ito et al., 2014), using a gene targeting strategy, the lung’s transcriptomic study has not been reported yet. - In this study, we used CRISPR-Cas9 technology to generate a global knock-out mouse model of the Bex2 gene. - The relative mRNA expression level analysis by RT-qPCR of the knocked-out mouse brain and lung indicated that the gene is successfully deleted. - Surprisingly, most of the enrichment is related to the cardiac system. - Similar results were obtained by other reserachers also (Yee et al., 2018).. - BEX2 is differentially expressed in breast tumors (Naderi et al., 2007), acute myeloid leukemia, and an increased expression was found in the MLL subtype (Rohrs et al., 2009). - Lungs are not only the respiratory organs, but there are pieces of evidence that they also play a role in immunity (Lefrançais et al., 2017). - We found that most of the KEGG pathways enriched in the immune system.. - Retnlg (DEG, log 2 = 1.81) is most likely expressed in nonadipose tissues (Lefrançais et al., 2017), indicates its possible role in the inflammation. - CSF3, otherwise identified as G-CSF, exists within the primary 17q12-21 asthma predisposition and exacerbation locus (Bisgaard et al., 2009). - Csf3r found in our upregulated DEGs (log 2 = 1.96) following the results of other researchers (Wang et al., 2019), may play a role in asthma. - Similarly, the downregulated genes are comparable to the results of the other researchers. - For instance, Myh6 among downregulated genes (log is following the results of other researchers (Yee et al., 2018), where they found that neonatal hyperoxia reduced the gene expression involved in contractile function (like Myl4, Myl7, Myh6, Myh7) etc. - BEX2 has been shown that it activates the NF-kB signaling pathway in breast cancer cells (Naderi et al., 2007), promotes the human glioblastoma cells proliferation via the NF-κB signaling pathway (Meng et al., 2014). - In this study, NF-κB is one of the prominent enriched pathway. - The most enriched gene among them is Tnf, which plays a central role in the development of the immune system. - Similarly, Bcl2a1d, from NF-kB pathway is related to apoptotic process (Cartagena et al., 2013). - Bdkrb2, log 2 = 1.62, has a functional role related to reactive oxygen species (Perhal et al., 2019).. - A recent study explains the role of BEX2 gene in allergic airway inflammation (Kim et al., 2020).. - In cell lines of leukemia, BEX2 was found to be expressed in MLLmu AML (Quentmeier et al., 2005).. - Besides, several DEGs identified were involved in the endocrine system and metabolic pathways. - RNA-Seq qPCR. - the lungs also play roles in the immune system and maybe a possible site for platelets’ biogenesis. - Adlat S, Sah RK, Hayel F, Chen Y, Bah FB et al. - Characterization of the Bex gene family in humans, mice, and rats. - Ashburner M, Ball CA, Blake JA, Botstein D, Butler H et al. - Bisgaard H, Bønnelykke K, Sleiman PM, Brasholt M, Chawes B et al.. - Cong L, Ran FA, Cox D, Lin S, Barretto R et al. - Foltz G, Ryu GY, Yoon JG, Nelson T, Fahey J et al. - Han C, Liu H, Liu J, Yin K, Xie Y et al. - Ito K, Yamazaki S, Yamamoto R, Tajima Y, Yanagida A et al. - Kim SD, Kang SA, Kim YW, Yu HS, Cho KS et al. - Kumar D, Bansal G, Narang A, Basak T, Abbas T et al. - Lefrançais E, Ortiz-Muñoz G, Caudrillier A, Mallavia B, Liu F et al.. - 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