Investigating the role of a lncRNA (4833427F10Rik) in mouse placental development and function
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Long non-coding RNAs (lncRNAs) have traditionally been viewed as transcriptional noise. However, experimental evidence of their cell-type-specific expression, presence of functional genetic elements, along with their role in developmental disorders, including reproductive disorders such as preeclampsia and intrauterine growth restriction (IUGR) have motivated us to investigate the role of candidate lncRNAs in placental development. Our hypothesis is that lncRNAs that have placental-specific expression will be important for proper placental development and function, and if dysregulated will result in perturbed placental/fetal development. Transcriptome analysis previously performed in our laboratory had identified [greater than] 1000 lncRNAs that were differentially expressed between the embryonic and extraembryonic tissues of E7.5 mouse conceptus. Specifically, a candidate lncRNA 4833427F10Rik had expression specific to the placental lineages in conceptus during early development stage. Both RT-qPCR and in situ hybridization verified the RNA-seq results and confirmed that 4833427F10Rik had expression specific to the placental lineage as early as E7.5 conceptus and had a sustained expression in the placenta throughout gestation. In this study, RNAscope was performed to investigate the expression of 4833427F10Rik in mouse conceptus. Results showed that in the conceptus, 4833427F10Rik had expression specific to the placenta lineage tissues but not in the embryo/fetus. To investigate the function of 4833427F10Rik, knock-out mouse lines were generated by removing the proximal promoter and part of exon 1 region of this lncRNA from the mouse genome using CRISPR/Cpf1 system with two single guide RNAs (sgRNAs). Based on the results from two knock-out mouse lines (line 28 and line 32), 4833427F10Rik knock-out was not embryonic lethal and did not alter the fetal growth (as measured by weights) during the early development of the knock-out mouse lines (3-week-old to 6-week-old). However, RT-PCR and RNAscope in the knock-out line showed that the knock-out mouse line 28 still had 4833427F10Rik expression in the E12.5 mouse placenta. Our finding about lncRNA 4833427F10Rik knock-out mice, therefore, might not be conclusive. However, the proximal promoter and the exon 1 region of mouse line 28 were ablated, and compared to wild-type mice, a larger maternal decidua and a smaller fetal placenta were seen by immunostaining in the homozygous mouse line 28, but this phenotype will need to be verified by additional experimentation. The expression levels as well as the resulting phenotype in another knock-out mouse line 32 will need to be explored in the future. In addition to generating knock-out mouse lines, we attempted to generate a Credriver mouse by knocking-in the Cre into exon 1 of the lncRNA 4833427F10Rik. The resulting Cre-driver mice could be leveraged for investigating spatioxiv temporal expression of the 4833427F10Rik, and for generating placental-specific knock-out of other candidate genes. However, two missense mutations (p.Ser305Leu, p.Asn327Tyr) were identified in the knock-in Cre sequence by Sanger sequencing, which may or may not affect the Cre enzyme folding or activity. Additional experiments will be needed to be performed to validate the Cre activity in the knock-in mice line in the future.
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M.S.