Project description:In order to determine the effect of neoR gene in Q175 locus, mRNASeq was performed to compare the transcriptional signatures of zQ175 knock-in neo-in (z_Q175 KI) (CHDI-81003003) and zQ175 KI neo-out (Z_Q175 (neo -) KI) (CHDI-81003019) mouse lines. The z_Q175 KI is a knock-in mouse line with humanized exon 1 (190-200 pure CAG repeats) derived from Q140 KI colony. It contains floxed neo cassette upstream of exon 1. The Z_Q175 KI (neo -) KI was generated by crossing the Z-Q175 KI line with a zp3-cre transgenic line to delete out the neo cassette.
Project description:In order to determine the effect of neoR gene in Q175 locus, miRNASeq was performed to compare the transcriptional signatures of zQ175 knock-in neo-in (z_Q175 KI) (CHDI-81003003) and zQ175 KI neo-out (Z_Q175 (neo -) KI) (CHDI-81003019) mouse lines. The z_Q175 KI is a knock-in mouse line with humanized exon 1 (190-200 pure CAG repeats) derived from Q140 KI colony. It contains floxed neo cassette upstream of exon 1. The Z_Q175 KI (neo -) KI was generated by crossing the Z-Q175 KI line with a zp3-cre transgenic line to delete out the neo cassette.
Project description:Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1~12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13~16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ~60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment.
Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a NeoR-based retrotransposition cassette. Cells were selected by G418 and used to prepare ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of G418-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016).
Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a neomycin-resistance-based retrotransposition cassette, as well as a hygromycin-resistance cassette on the plasmid backbone. For this set of experiments, cells were only selected for transfection (hygromycin) but not for retrotransposition (neomycin). Then we prepared ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of hygromycin-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016). For some libraries, the linker-ligated genomic DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to limit amplification from the plasmid and enrich for retrotransposition-mediated insertion events into the genomic DNA.
Project description:GRTH/DDX 25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. Regulation of spermatogenesis occurs at the levels of transcription, post-transcriptional and translational levels which needs to be explored in detail. miRNAs regulate the expression of important genes involved in spermatid elongation process. Differential miRNA expression in round spermatids (RS) of GRTH KO and GRTH-KI mice in comparison with mRNAs remains unexplored. Our miRNAseq analysis of small RNAs obtained from RS reveal differential expression of important miRNAs that regulate spermatid differentiation, apoptosis, chromatin compaction and ubiquitination. mRNAseq analysis of RS isolated from GRTH-KI and GRTH-KO mice models were also carried-out to study the putative miRNA targets and mRNA-miRNA interaction. mRNAseq analysis revealed specific set of mRNAs that are critical for sperm chromatin compaction, ubiquitination and spermatid elongation etc. miRNA-mRNA interaction prediction highlighted differentially expressed/enriched miRNA transcripts whose predicted mRNA targets were differentially expressed. Based on the interaction information of the miRNAs-mRNAs differential analysis, the miRNA-mRNA network was constructed. These results demonstrate the importance of miRNA in the translational arrest and stability of germ cell specific mRNAs which are critical for later stages of spermiogenesis and fertility.