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 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: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: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:Analysis of gene expression in leukocytes taken from 13 participants 1 week before or after the skydive (baseline), before boarding the airplane (pre-boarding), at landing and 1 h after the parachute jump (1 h landing). Results identify a subset of genes expressed by natural killer cells that are upregulated in leukocytes in response to acute psychological stress.
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:The c-myc proto-oncogene is activated by translocation in Burkitt's lymphoma and substitutions in codon 58 stabilize the Myc protein or augment its oncogenic potential. In wild-type Myc, phosphorylation of Ser 62 and Thr 58 provide a landing pad for the peptidyl prolyl-isomerase Pin1, which in turn promotes Ser 62 dephosphorylation and Myc degradation. However, the role of Pin1 in Myc-induced lymphomagenesis remains unknown. We show here that genetic ablation of Pin1 reduces lymphomagenesis in Eµ-myc transgenic mice. In both Pin1-deficient B-cells and MEFs, the proliferative response to Myc was selectively impaired, with no alterations in Myc-induced apoptosis or mitogen-induced cell cycle entry. This proliferative defect wasn't attributable to alterations in either Ser 62 phosphorylation or Myc-regulated transcription, but to the indirect activation of an Arf-p53 dependent cytostatic response. Pin1 silencing in lymphomas retarded disease progression in mice, making Pin1 an attractive therapeutic target in Myc-driven tumors. RNAseq samples of Pin1+/+ control (n=4), Pin1 -/- control (n=2), Pin1+/+ Eµ-myc pre-tumoral (n=3), and Pin1-/- Eµ-myc pre-tumoral (n=4) B cells.