Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors.
2017; 14 (12): 1205–12
Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.
View details for PubMedID 29106405
- Comprehensive Cell Type Specific Transcriptomics of the Human Kidney bioRxiv 2017
Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.
2015; 16 (7): 718–28
Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α(+) (CD103(+)) cDC1 lineage, and the CD11b(+) cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H(-)Ly6C(-) pre-DCs) or cDC2 lineage (Siglec-H(-)Ly6C(+) pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.
View details for DOI 10.1038/ni.3200
View details for PubMedID 26054720
Klhl14 Antisense RNA is a Target of Key Skeletogenic Transcription Factors in the Developing Intervertebral Disc.
2019; 44 (5): E260–E268
RNA in situ hybridization (RISH) allows for validation and characterization of the long noncoding (lnc) natural antisense RNA (NAT) Klhl14as in the embryonic murine intervertebral disc (IVD) in the context of loss-of-function mutants for key transcription factors (TFs) in axial skeleton development.Validation of Klhl14as in the developing murine IVD.The IVD is a focus of regenerative medicine; however, processes and signaling cascades resulting in the different cell types in a mature IVD still require clarification in most animals including humans. Technological advances increasingly point to implications of lnc NATs in transcription/translation regulation. Transcriptome data generation and analysis identified a protein encoding transcript and related noncoding antisense transcript as downregulated in embryos devoid of key TFs during axial skeleton development. Here, primarily, the antisense transcript is analyzed in this loss-of-function context.4930426D05Rik and 6330403N15Rik were identified as Klhl14as and sense, respectively, two transcripts downregulated in the vertebral column of midgestation Pax1 and Pax9 mutant mouse embryos. RISH on wildtype and mutant embryos for the TF encoding genes Pax1/Pax9, Sox5/Sox6/Sox9, and Bapx1 was used to further analyze Klhl14as in the developing IVD.Klhl14as and Klhl14 were the top downregulated transcripts in Pax1; Pax9 E12.5 embryos. Our data demonstrate expression of Klhl14as and sense transcripts in the annulus fibrosus (AF) and notochord of the developing IVD. Klhl14as expression in the inner annulus fibrosus (iAF) seems dependent on the TFs Pax1/Pax9, Sox6, Sox9, and Bapx1.We are the first to suggest a role for the lncRNA Klhl14as in the developing IVD. Our data link Klhl14as to a previously established gene regulatory network during axial skeleton development and contribute further evidence that lnc NATs are involved in crucial gene regulatory networks in eukaryotic cells.N/A.
View details for DOI 10.1097/BRS.0000000000002827
View details for PubMedID 30086079
Regulatory Functions of Pax1 and Pax9 in Mammalian Cells
Gene Expression and Regulation in Mammalian Cells-Transcription Toward the Establishment of Novel Therapeutics
InTechOpen. 2018; 1: 181–207
View details for DOI 10.5772/intechopen.71920
Single-cell transcriptomes identify human islet cell signatures and reveal cell-type-specific expression changes in type 2 diabetes.
2017; 27 (2): 208–22
Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type-specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis.
View details for DOI 10.1101/gr.212720.116
View details for PubMedID 27864352
View details for PubMedCentralID PMC5287227
An Integrative Developmental Genomics and Systems Biology Approach to Identify an In Vivo Sox Trio-Mediated Gene Regulatory Network in Murine Embryos.
BioMed research international
2017; 2017: 8932583
Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis.
View details for DOI 10.1155/2017/8932583
View details for PubMedID 28630873
View details for PubMedCentralID PMC5467288
A developmental transcriptomic analysis of Pax1 and Pax9 in embryonic intervertebral disc development.
2017; 6 (2): 187–99
Pax1 and Pax9 play redundant, synergistic functions in the patterning and differentiation of the sclerotomal cells that give rise to the vertebral bodies and intervertebral discs (IVD) of the axial skeleton. They are conserved in mice and humans, whereby mutation/deficiency of human PAX1/PAX9 has been associated with kyphoscoliosis. By combining cell-type-specific transcriptome and ChIP-sequencing data, we identified the roles of Pax1/Pax9 in cell proliferation, cartilage development and collagen fibrillogenesis, which are vital in early IVD morphogenesis. Pax1 is up-regulated in the absence of Pax9, while Pax9 is unaffected by the loss of Pax1/Pax9 We identified the targets compensated by a single- or double-copy of Pax9 They positively regulate many of the cartilage genes known to be regulated by Sox5/Sox6/Sox9 and are connected to Sox5/Sox6 by a negative feedback loop. Pax1/Pax9 are intertwined with BMP and TGF-B pathways and we propose they initiate expression of chondrogenic genes during early IVD differentiation and subsequently become restricted to the outer annulus by the negative feedback mechanism. Our findings highlight how early IVD development is regulated spatio-temporally and have implications for understanding kyphoscoliosis.
View details for DOI 10.1242/bio.023218
View details for PubMedID 28011632
View details for PubMedCentralID PMC5312110
Genome wide binding (ChIP-Seq) of murine Bapx1 and Sox9 proteins in vivo and in vitro.
2016; 10: 51–53
This work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq) of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs . Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null) was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue.
View details for DOI 10.1016/j.gdata.2016.09.002
View details for PubMedID 27672560
View details for PubMedCentralID PMC5030313
Gene expression profiles of Bapx1 expressing FACS sorted cells from wildtype and Bapx1-EGFP null mouse embryos.
2015; 5: 103–5
The data described in this article refers to Chatterjee et al. (2015) "In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column" (GEO GSE35649) . Transcriptional profiling combined with genome wide binding data is a powerful tool to elucidate the molecular mechanism behind vertebrate organogenesis. It also helps to uncover multiple roles of a single gene in different organs. In the above mentioned report we reveal the function of the homeobox gene Bapx1 during the embryogenesis of five distinct organs (vertebral column, spleen, gut, forelimb and hindlimb) at a relevant developmental stage (E12.5), microarray analysis of isolated wildtype and mutant cells in is compared in conjunction with ChIP-Seq analysis. We also analyzed the development of the vertebral column by comparing microarray and ChIP-Seq data for Bapx1 with similarly generated data sets for Sox9 to generate a gene regulatory network controlling various facets of the organogenesis.
View details for DOI 10.1016/j.gdata.2015.05.031
View details for PubMedID 26101748
View details for PubMedCentralID PMC4474491
Pleiotropic functions for transcription factor zscan10.
2014; 9 (8): e104568
The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in in vitro assays. Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof.
View details for DOI 10.1371/journal.pone.0104568
View details for PubMedID 25111779
View details for PubMedCentralID PMC4128777
Generating mouse lines for lineage tracing and knockout studies.
Methods in molecular biology (Clifton, N.J.)
2014; 1194: 37–62
In 2007 Capecchi, Evans, and Smithies received the Nobel Prize in recognition for discovering the principles for introducing specific gene modifications in mice via embryonic stem cells, a technology, which has revolutionized the field of biomedical science allowing for the generation of genetically engineered animals. Here we describe detailed protocols based on and developed from these ground-breaking discoveries, allowing for the modification of genes not only to create mutations to study gene function but additionally to modify genes with fluorescent markers, thus permitting the isolation of specific rare wild-type and mutant cell types for further detailed analysis at the biochemical, pathological, and genomic levels.
View details for DOI 10.1007/978-1-4939-1215-5_3
View details for PubMedID 25064097
In vivo genome-wide analysis of multiple tissues identifies gene regulatory networks, novel functions and downstream regulatory genes for Bapx1 and its co-regulation with Sox9 in the mammalian vertebral column.
2014; 15: 1072
Vertebrate organogenesis is a highly complex process involving sequential cascades of transcription factor activation or repression. Interestingly a single developmental control gene can occasionally be essential for the morphogenesis and differentiation of tissues and organs arising from vastly disparate embryological lineages.Here we elucidated the role of the mammalian homeobox gene Bapx1 during the embryogenesis of five distinct organs at E12.5 - vertebral column, spleen, gut, forelimb and hindlimb - using expression profiling of sorted wildtype and mutant cells combined with genome wide binding site analysis. Furthermore we analyzed the development of the vertebral column at the molecular level by combining transcriptional profiling and genome wide binding data for Bapx1 with similarly generated data sets for Sox9 to assemble a detailed gene regulatory network revealing genes previously not reported to be controlled by either of these two transcription factors.The gene regulatory network appears to control cell fate decisions and morphogenesis in the vertebral column along with the prevention of premature chondrocyte differentiation thus providing a detailed molecular view of vertebral column development.
View details for DOI 10.1186/1471-2164-15-1072
View details for PubMedID 25480362
View details for PubMedCentralID PMC4302147
Making sense of Dlx1 antisense RNA.
2013; 376 (2): 224–35
Long non-coding RNAs (lncRNAs) have been recently recognized as a major class of regulators in mammalian systems. LncRNAs function by diverse and heterogeneous mechanisms in gene regulation, and are key contributors to development, neurological disorders, and cancer. This emerging importance of lncRNAs, along with recent reports of a functional lncRNA encoded by the mouse Dlx5-Dlx6 locus, led us to interrogate the biological significance of another distal-less antisense lncRNA, the previously uncharacterized Dlx1 antisense (Dlx1as) transcript. We have functionally ablated this antisense RNA via a highly customized gene targeting approach in vivo. Mice devoid of Dlx1as RNA are viable and fertile, and display a mild skeletal and neurological phenotype reminiscent of a Dlx1 gain-of function phenotype, suggesting a role for this non-coding antisense RNA in modulating Dlx1 transcript levels and stability. The reciprocal relationship between Dlx1as and Dlx1 places this sense-antisense pair into a growing class of mammalian lncRNA-mRNA pairs characterized by inverse regulation.
View details for DOI 10.1016/j.ydbio.2013.01.035
View details for PubMedID 23415800
A conditional mouse line for lineage tracing of Sox9 loss-of-function cells using enhanced green fluorescent protein.
2013; 35 (12): 1991–96
Traditionally, conditional knockout studies in mouse have utilized the Cre or Flpe technology to activate the expression of reporter genes such as lacZ or PLAP. Employing these reporter genes, however, requires tissue fixation. To make way for downstream in vivo or in vitro applications, we have inserted enhanced green fluorescent protein (EGFP) into the endogenous Sox9 locus and generated a novel conditional Sox9 null allele, by flanking the entire Sox9 coding region with loxP sites and inserting an EGFP reporter gene into the 3'-UTR allowing for EGFP to be expressed upon Sox9 loss of function yet under the control of the endogenous Sox9 promoter. Mating this new allele to any Cre-expressing line, the fate of Sox9 null cells can be traced in the cell type of interest in vivo or in vitro after fluorescence-activated cell sorting.
View details for DOI 10.1007/s10529-013-1303-6
View details for PubMedID 23907671
Stemming the Degeneration: IVD Stem Cells and Stem Cell Regenerative Therapy for Degenerative Disc Disease.
Advances in stem cells
The intervertebral disc (IVD) is immensely important for the integrity of vertebral column function. The highly specialized IVD functions to confer flexibility and tensile strength to the spine and endures various types of biomechanical force. Degenerative disc disease (DDD) is a prevalent musculoskeletal disorder and is the major cause of low back pain and includes the more severe degenerative lumbar scoliosis, disc herniation and spinal stenosis. DDD is a multifactorial disorder whereby an imbalance of anabolic and catabolic factors, or alterations to cellular composition, or biophysical stimuli and genetic background can all play a role in its genesis. However, our comprehension of IVD formation and theetiology of disc degeneration (DD) are far from being complete, hampering efforts to formulate appropriate therapies to tackle DD. Knowledge of the stem cells and various techniques to manipulate and direct them to particular fates have been promising in adopting a stem-cell based regenerative approach to DD. Moreover, new evidence on the residence of stem/progenitor cells within particular IVD niches has emerged holding promise for future therapeutic applications. Existing issues pertaining to current therapeutic approaches are also covered in this review.
View details for DOI 10.5171/2013.724547
View details for PubMedID 23951558
View details for PubMedCentralID PMC3742038
Pax1(EGFP): new wildtype and mutant EGFP mouse lines for molecular and fate mapping studies.
Genesis (New York, N.Y. : 2000)
2013; 51 (6): 420–29
The Paired box gene 1 (Pax1) transcription factor plays essential roles in the development of axial skeleton, scapula, pelvic girdle, and thymus. Delineating its pleiotropic and molecular roles in the various tissues requires the ability to track and isolate the Pax1-expressing cells for downstream high-throughput experiments such as microarray and RNA-sequencing. With these applications in mind, we have generated two new mouse lines-a Pax1 wildtype (WT) mouse line that co-expresses enhanced green fluorescent protein (EGFP) with functional Pax1, and a Pax1 knockout mouse line which expresses EGFP under the control of Pax1 promoter, using the internal ribosome entry site (IRES) and 2A-peptide multi-cistron concatenating strategies. These mouse lines facilitate the isolation and enrichment of Pax1-specific cells from Pax1-positive and Pax1-null embryos using fluorescence activated cell sorting (FACS). They can be also be used in parallel to investigate the stage- and tissue-specific molecular functions of Pax1.
View details for DOI 10.1002/dvg.22379
View details for PubMedID 23377878
Bridging the Gap: Understanding Embryonic Intervertebral Disc Development.
Cell & developmental biology
2012; 1 (2)
The intervertebral disc (IVD) is a multi-component structure consisting of a heterogeneous population of cells that form the central nucleus pulposus, encased by the fibrous annulus fibrosus and the cartilage end-plate. The essential function of the IVD is to withstand biomechanical forces, confer tensile strength and flexibility in motion to the spine. Disc degenerative disease (DD) is a prevalent ailment that affects the general population, often manifesting either in the form of lower back pain or as deformities of the spine such as degenerative lumbar scoliosis or in severe cases as disc herniation. With the aid of mutant mouse models generated through traditional knock-out strategies and spontaneous mutants, scientists have been able to elucidate some of the fundamental mechanisms of embryonic IVD development. Mutual interaction between the notochord and vertebral bodies are instrumental in the proper formation of the IVD. In this review, the known and proposed molecular mechanisms underlying these processes and the areas that require further investigation are discussed. Sufficient knowledge on the molecular mechanisms of IVD formation and the etiology of IVD degeneration is currently lacking and this has greatly hampered efforts to design appropriate and effective therapies for DD. With the dawn of the next-generation sequencing and better tools to engineer the genome, elucidation of the mechanism of IVD formation and the molecular basis of the pathology of DD ought to be an appealing avenue for researchers to pursue.
View details for PubMedID 23106046
View details for PubMedCentralID PMC3481539
Mouse strain specific gene expression differences for illumina microarray expression profiling in embryos.
BMC research notes
2012; 5: 232
In the field of mouse genetics the advent of technologies like microarray based expression profiling dramatically increased data availability and sensitivity, yet these advanced methods are often vulnerable to the unavoidable heterogeneity of in vivo material and might therefore reflect differentially expressed genes between mouse strains of no relevance to a targeted experiment. The aim of this study was not to elaborate on the usefulness of microarray analysis in general, but to expand our knowledge regarding this potential "background noise" for the widely used Illumina microarray platform surpassing existing data which focused primarily on the adult sensory and nervous system, by analyzing patterns of gene expression at different embryonic stages using wild type strains and modern transgenic models of often non-isogenic backgrounds.Wild type embryos of 11 mouse strains commonly used in transgenic and molecular genetic studies at three developmental time points were subjected to Illumina microarray expression profiling in a strain-by-strain comparison. Our data robustly reflects known gene expression patterns during mid-gestation development. Decreasing diversity of the input tissue and/or increasing strain diversity raised the sensitivity of the array towards the genetic background. Consistent strain sensitivity of some probes was attributed to genetic polymorphisms or probe design related artifacts.Our study provides an extensive reference list of gene expression profiling background noise of value to anyone in the field of developmental biology and transgenic research performing microarray expression profiling with the widely used Illumina microarray platform. Probes identified as strain specific background noise further allow for microarray expression profiling on its own to be a valuable tool for establishing genealogies of mouse inbred strains.
View details for DOI 10.1186/1756-0500-5-232
View details for PubMedID 22583621
View details for PubMedCentralID PMC3497855
New Bapx1(Cre-EGFP) mouse lines for lineage tracing and conditional knockout studies.
Genesis (New York, N.Y. : 2000)
2012; 50 (4): 375–83
To gain insight into the roles of various genes in development and to circumvent embryonic lethality that hinders genetic studies, lineage tracing and conditional knockout techniques have been widely performed on mice using the increasing numbers of gene-targeted Cre mouse lines. Employing the internal ribosome entry site (IRES) and the 2A peptide multicistronic expression strategies, we report two new Bapx1 mouse lines with functional Bapx1 whereby Cre and enhanced green fluorescence protein (EGFP) are expressed discretely under the control of the Bapx1 promoter. These mouse lines, when mated with the Rosa26R-lacZ reporter line, can be used to trace the lineage of Bapx1-expressing cells whereas stage-specific, spatial expression of Bapx1 can be visualized by the EGFP fluorescence. In addition, both of our Bapx1(Cre-EGFP) mouse lines can be used to enrich for Bapx1-specific cells and also serve as effective conditional knockout tools to investigate gene functions in the skeleton and/or visceral organs.
View details for DOI 10.1002/dvg.20802
View details for PubMedID 21913311
Making no bones about it: Transcription factors in vertebrate skeletogenesis and disease.
Trends in developmental biology
2012; 6: 45–52
Skeletogenesis is a complex multi-step process, which involves many genes and pathways. The tightly regulated interplay between these genes in these pathways ensures a correct and timely organogenesis and it is imperative that we have a fair understanding of the major genes and gene families involved in the process. This review aims to give a deeper insight into the roles of 3 major transcription factor families involved in skeleton formation: Sox, Runx and Pax and to look at the human skeleotogenic phenotypes associated with mutations in these genes.
View details for PubMedID 23950621
View details for PubMedCentralID PMC3742036
Generation of mice with a novel conditional null allele of the Sox9 gene.
2011; 33 (8): 1551–58
Sox9 is expressed in multiple tissues during mouse development and adulthood. Mutations in the Sox9 gene or changes in expression levels can be attributed to many congenital diseases. Heterozygous loss-of-function mutations in the human SOX9 gene cause Campomelic dysplasia, a semi-lethal skeletal malformation syndrome. Disruption of Sox9 by conventional gene targeting leads to perinatal lethality in heterozygous mice, hence hampering the feasibility to obtain the homozygous Sox9 null mice for in vivo functional studies. In this study, we generated a conditional allele of Sox9 (Sox9 ( tm4.Tlu )) by flanking exon 1 with loxP sites. Homozygous mice for the Sox9 ( tm4.Tlu ) allele (Sox9 ( flox/flox )) are viable, fertile and indistinguishable from wildtype (WT) mice, indicating that the Sox9 ( tm4.Tlu ) allele is a fully functional Sox9 allele. Furthermore, we demonstrated that Cre-mediated recombination using a Col2a1-Cre line resulted in specific ablation of Sox9 activity in cartilage tissues.
View details for DOI 10.1007/s10529-011-0608-6
View details for PubMedID 21484342
Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines.
2011; 6 (12): e28885
Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9(IRES-EGFP)) or the F2A (Sox9(F2A-EGFP)) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome 'skipping' mechanism. To investigate if the discrepancy in the 'skipping' process was locus-dependent, we further analyzed the FLAG(3)-Bapx1(F2A-EGFP) mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the 'skipping' mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1(F2A-Cre-F2A-EGFP)). While the 'skipping' was highly efficient between Bapx1 and Cre, the 'skipping' between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the 'skipping' at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.
View details for DOI 10.1371/journal.pone.0028885
View details for PubMedID 22216134
View details for PubMedCentralID PMC3244433
Nuclear accumulation of an uncapped RNA produced by Drosha cleavage of a transcript encoding miR-10b and HOXD4.
2011; 6 (10): e25689
Patterning of the animal embryo's antero-posterior (AP) axis is dependent on spatially and temporally regulated Hox gene expression. The murine Hoxd4 gene has been proposed to harbour two promoters, an upstream promoter P2, and a downstream promoter P1, that lie 5.2 and 1.1 kilobase pairs (kb) upstream of the coding region respectively. The evolutionarily conserved microRNA-10b (miR-10b) gene lies in the Hoxd4 genomic locus in the intron separating the non-coding exons 4 and 5 of the P2 transcript and directly adjacent to the proposed P1 promoter. Hoxd4 transcription is regulated by a 3' neural enhancer that harbours a retinoic acid response element (RARE). Here, we show that the expression profiles of Hoxd4 and miR-10b transcripts during neural differentiation of mouse embryonal carcinoma (EC) P19 cells are co-ordinately regulated, suggesting that both Hoxd4 and miR-10b expression is governed by the neural enhancer. Our observation that P1 transcripts are uncapped, together with the mapping of their 5' ends, strongly suggests that they are generated by Drosha cleavage of P2 transcripts rather than by transcriptional initiation. This is supported by the colocalization of P1 and P2 transcripts to the same posterior expression domain in the mouse embryo. These uncapped P1 transcripts do not appear to possess an Internal Ribosomal Entry Site (IRES), but accumulate within multiple punctate bodies within the nucleus suggesting that they play a functional role. Finally, similar uncapped Drosha-cleaved P1-like transcripts originating from the paralogous Hoxb4/miR-10a locus were also identified. We propose that these transcripts may belong to a novel class of regulatory RNAs.
View details for DOI 10.1371/journal.pone.0025689
View details for PubMedID 21991333
View details for PubMedCentralID PMC3185001