Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

ABSTRACT: 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.

ORGANISM(S): synthetic construct

PROVIDER: GSE231138 | GEO | 2023/05/05

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

An Improved Platform for Functional Assessment of Large Protein Libraries in Mammalian Cells

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.

2019-09-30 | GSE135717 | GEO

Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

Project description:Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

| PRJNA962992 | ENA

Project description:Landing pad resequencing of Sf9 cells

| PRJNA942085 | ENA

Optimized metrics for combinatorial and orthogonal CRISPR screens

Project description:CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a system (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.

2023-05-10 | GSE215175 | GEO

Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

Project description:Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by the introduction of the transcription factors Oct4, Sox2, Klf4 and cMyc using various methods. Here, we describe a new approach for the derivation of murine iPSCs using a polycistronic non-viral inducible vector integrated into pseudo attP sites via the C31 integrase-mediated site-specific recombination and subsequent vector excision by Cre recombinase. The pluripotency of the derived iPSCs was proved by in vitro and in vivo tests. The derived transgene-free iPSCs reactivated the endogenous pluripotency genes like e.g. Oct4, Sox2 and Nanog and the global gene expression profiles of iPSCs lines are highly similar to ESCs and distinct from parental murine fibroblasts. We demonstrated the differentiation potential of iPSCs by generation cells of the three germ layers as well as we successfully created germline chimeric mice from transgene-free iPSCs. In this study, we presented an efficient method for the generation of transgene-free iPSCs using dual-recombinase technology.

2014-03-29 | GSE51605 | GEO

DNA sequencing to validate random deletions in Mycoplasma pneumoniae

Project description:We present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of Lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.

2021-08-18 | E-MTAB-9582 | biostudies-arrayexpress

Transposon sequencing to identify random insertions of lox sites in Mycoplasma pneumoniae

Project description:This dataset was used to assess the random insertion by tranposases of lox sites in Mycoplasma pneumoniae. This is part of the protocol LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of Lox sites by transposon mutagenesis, and the generation of mutants via cre recombinase, catalogued via deep-sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from >50 bp to 28 Kb, which represent 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other similar regions that could not, providing a guide for future genome reductions.

2021-08-18 | E-MTAB-9590 | biostudies-arrayexpress

Simple Derivation of Transgene-Free iPS Cells by a Dual Recombinase Approach

2014-03-29 | E-GEOD-51605 | biostudies-arrayexpress

A Proximity Labeling-based Orthogonal Trap Approach for Screening HDAC8 substrates

Project description:We report a proximity labeling-based orthogonal trap approach for investigation of HDAC8 substrates in living cells. In this strategy, the genetic fusions that coupled the engineering enzyme APEX2 with HDAC8 are designed to trap substrate proteins in cellular context, by which they contribute to the covalent tagging of the transient neighboring binders. The tagged proteins are then employed with an integrated orthogonal enrichment strategy that allows for direct identification of acetylated sites on targets.

2022-06-17 | PXD034616 |

Influence of cell permeable Tre-recombinase (CPTR) treatment on T-cell gene expression

Project description:CEM-ss cells were treated with or without a recombinantly expressed recombinase and gene expression profiles were compared.

2012-02-22 | GSE32643 | GEO