Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

Conditional gene expression is a powerful tool for genetic analysis of biological phenomena. In the widely used “lox-stop-lox” approach, insertion of a stop cassette consisting of a series of stop codons and polyadenylation signals flanked by lox sites into the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been successfully used in many experimental systems, this design has certain limitations. Here, we describe the Floxed exon (Flexon) approach, which uses a stop cassette composed of an artificial exon flanked by artificial introns, designed to cause premature termination of translation and nonsense-mediated decay of the mRNA and allowing for flexible placement into a gene. We demonstrate its efficacy in Caenorhabditis elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression of green fluorescent protein (GFP) is obtained in specific lineages. We also demonstrate its efficacy in an endogenous gene context: we inserted a flexon into the Argonaute gene rde-1 to abrogate RNA interference (RNAi), and restored RNAi tissue specifically by expression of Cre. Finally, we describe several potential additional applications of the Flexon approach, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation, and generation of genetic mosaics. The Flexon approach should be feasible in any system where a site-specific recombination-based method may be applied.

A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.

A toolkit to generate inducible and interconvertible Drosophila transgenes

ABSTRACTThe existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a multipurpose modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of transgenic fly lines.

Postnatal inactivation of Shank3 expression is insufficient to produce behavioral phenotypes associated with germline deficiency

Background: The developmental origins of Autism Spectrum Disorder (ASD) remain largely unknown. To begin to answer this question, a few studies have implemented conditional gene expression technology to induce or rescue ASD-causing mutations at different times during mouse development. Methods: In the current study, we crossed a ubiquitously-expressed inducible Cre line with mice containing loxP sites surrounding Shank3 exons 4-22 (Shank3 e4-22flox). We then delivered tamoxifen to determine the effects of disrupting Shank3 expression during two different time periods in development. Results: We found that reducing Shank3 expression in fully developed mice had minimal effects on behaviors that are characteristic of the conventional knockout mice. Similarly, disrupting Shank3 expression during an earlier time period (postnatal day 10-21) did not induce behaviors associated with germline deficiency. However, we observed differences in electrophysiological phenotypes for the two groups of conditional knockout mice. Conclusions: Because inducing mutations in adult mice or during early postnatal development is insufficient to cause ASD-like behaviors, Shank3 may play a role even earlier in development, but limited efficiency of the disruption could have contributed to our negative results. We also report considerable mortality when delivering tamoxifen across several different routes of administration, bringing further attention to caution the use of this type of conditional gene expression technology in future studies.

Presentation of the Molecular Subtypes of Breast Cancer Detected By Immunohistochemistry in Surgically Treated Patients

INTRODUCTION: The detection of estrogen, progesterone and HER-2 neu receptors on the surface of the tumour cell is a significant prognostic factor, alone or in combination. The presence or absence of receptors on the surface of the tumour cell is associated with the conditional gene expression in the tumour cell itself. Based on these genetically determined expressions of the tumour cell, five molecular subtypes of breast cancer have been classified on the St. Gallen International Expert Consensus in 2011 that can be immunohistochemically detected, with each subtype manifesting certain prognosis and aggression.AIM: Analyzing the presentation of molecular subtypes of breast cancer that are immunohistochemically detected in surgically treated patients at the Clinic for Thoracic and Vascular Surgery.MATERIAL AND METHODS: We used the international classification on molecular subtypes of breast cancer which divides them into: Luminal A (ER+ and/or PR+, HER-2 negative, Ki-67 < 14%), Luminal B with HER-2 negative (ER+ and/or PR+, HER-2 negative, Ki-67 ≥ 14%), Luminal B with HER-2 positive (ER+ and/or PR+, HER-2+, any Ki-67), HER-2 enriched (ER-, PR-, HER-2+), and basal-like (triple negative) (ER-, PR-, HER-2 negative, CK5/6+ and/or EGFR+). A total of 290 patients, surgically treated for breast cancer, were analysed during 2014.RESULTS: In our analysis, we found that Luminal A was present in 77 (26.55%) patients, Luminal B HER-2 negative was present in 91 (31.38%) patients, Luminal B HER-2 positive was present in 70 (24.14%) patients, HER-2 enriched was present in 25 (8.62%) patients and basal-like (or triple negative) was present in 27 (9.31%) patients.CONCLUSION: Detecting the subtype of breast cancer is important for evaluating the prognosis of the disease, but also for determining and providing an adequate therapy. Therefore, determining the subtype of breast cancer is necessary for the routine histopathological assay.