Project description:Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed, and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Finally, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.
Project description:The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered “landing pad” site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting, and insufficient for more complex cell-based assays for protein function. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable new avenues for mammalian synthetic biology. By designing plasmids with two recombinase sites, we demonstrate that we can avoid genomic integration of undesirable bacterial DNA elements. We also created “double landing pad” cells simultaneously harboring two orthogonal Bxb1 recombinase sites. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, opening up new functional readouts for large-scale functional assays.
Project description:To quantify the contribution of specific transcription factor binding versus chromatin context in chromatin opening, we inserted libraries containing hundreds of CREs into a landing pad within a neutral chromatin environment, devoid of activating or repressive chromatin modifications, in mESCs using Recombination-Mediated Cassette Exchange (RMCE). Chromatin accessibility of the inserted fragments was then profiled by targeted SMF using PCR primers that anneal to a synthetic flanking sequence, allowing unambiguous differentiation of the ectopic site from its endogenous counterpart. To investigate whether H3K27Ac contributes to chromatin accessibility at enhancers, we globally reduced H3K27Ac levels by chemically inhibiting the histone acetyltransferase p300 with the small molecule A-485 (24 hours treatment, final concentration: 3 μM). This was performed in the same mESCs carrying the CREs at the landing pad. The dataset generated includes amplicon-based SMF data from the ectopic site in DMSO- and A-485-treated conditions in mouse cells (i.e., TC-1 knock-out of the three DNA methyl transferases (DNMT TKO) mESCs). Two biological replicates were generated for each condition. In summary, cells were collected for SMF, which marks accessible cytosines via recombinant methyltransferases, followed by bisulfite sequencing to infer protein-DNA interactions and chromatin accessibility at single-molecule resolution. The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit and sequenced on an Illumina platform, using either a MiSeq 250 bp paired-end run, a MiSeq i100 250 bp paired-end run, or a NextSeq 2000 P1 300 bp paired-end run. Reads were pre-processed with TrimGalore and a custom R script was used to trim the plasmid backbone from the reads. After, pre-processed reads were aligned using QuasR. Further analyses were conducted using custom scripts available at https://github.com/Krebslabrep/TF-chromatin.git.
Project description:Effect of phenobarbital on Sf9 cell cultures genes expression. RNA from phenobarbital treated Sf9 cell cultures were compared to control treated (DMSO) Sf9 cell
Project description:To build therapeutic strains, Escherichia coli Nissle (EcN) have been engineered to express antibiotics, toxin-degrading enzymes, immunoregulators, and anti-cancer chemotherapies. For efficacy, the recombinant genes need to be highly expressed, but this imposes a burden on the cell, and plasmids are difficult to maintain in the body. To address these problems, we have developed landing pads in the EcN genome and genetic circuits to control therapeutic gene expression. These tools were applied to EcN SYNB1618, undergoing clinical trials as a phenylketonuria treatment. The pathway for converting phenylalanine to trans-cinnamic acid was moved to a landing pad under the control of a circuit that keeps the pathway off during storage. The resulting strain (EcN SYN8784) achieved higher activity than EcN SYNB1618, reaching levels near when the pathway is carried on a plasmid. This work demonstrates a simple system for engineering EcN that aids quantitative strain design for therapeutics.
Project description:published at http://dx.plos.org/10.1371/journal.pone.0025708 Effect of hormone agonists on Sf9 cells : methoxyfenozide (Mtfz) and methoprene (Mtp)
Project description:published at http://dx.plos.org/10.1371/journal.pone.0025708 Effect of hormone agonists on Sf9 cells : methoxyfenozide (Mtfz) and methoprene (Mtp) We have 12 microarrays corresponding to 6 dye swaps , there is 3 biological replicates for each comparison. 6 microarrays: dye swap of 3 biological replicates corresponding to the comparison between Sf9 cell lines treatment with methoprene (Mtp) versus DMSO control treatment. And 6 microarrays: dye swap of 3 biological replicates corresponding to the comparison between Sf9 cell lines treatment with methoxyfenozide(mtfz) versus DMSO control treatment
Project description:Transcription and RNA processing are tightly coupled and precisely coordinated to ensure appropriate levels of mature transcripts. The C-terminal domain (CTD) of RNA polymerase II (Pol II) is phosphorylated differentially during the transcription cycle and serves as a landing pad for a variety of transcriptional regulators and RNA processing proteins. PHD finger protein 3 (PHF3) binds to the serine-2 phosphorylated Pol II CTD with its Spen Paralogue and Orthologue C-terminal (SPOC) domain and regulates transcription elongation and mRNA stability. Here we show that PHF3 binds target RNAs by recognizing a G-rich motif prone to form G-quadruplexes (G4s). Two PHF3 zinc finger domains, PHD (plant homeo domain) and TLD (TFIIS-like domain) act in concert to bind and destabilize target RNAs and their deletion in HEK293T cells causes massive deregulation of gene expression. Together these results establish PHF3 as a Pol II and an RNA-binding protein that coordinates transcription elongation with RNA decay to regulate neuronal gene expression.