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 (Sox9IRES-EGFP) or the F2A (Sox9F2A-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 FLAG3-Bapx1F2A-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 (Bapx1F2A-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.

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.

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.

The long-standing traditional method of delivering embryonic stem (ES) cells adjacent to the inner cell mass (ICM) of blastocysts to generate chimeras improved with the advent of laser- or Piezo assisted 8-cell embryo microinjection. Building on this technology but omitting either the laser or the Piezo to penetrate the zona pellucida and making use of earlier embryonic stages (2-cell and 4-cell), we were able to significantly speed up and economize our ES cell microinjection and chimera production throughput. We demonstrate here that embryonic (ES) and induced pluripotent stem (iPS) cells can stay fully pluripotent when delivered into 2-cell- and 4-cell-stage embryos, long before they would naturally be incorporated into the ICM of a blastocyst (E3.5) and give rise to high percentage and germline transmitting chimeras.

BackgroundVertebrate 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.ResultsHere 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1072) contains supplementary material, which is available to authorized users.