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Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies

ABSTRACT: Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies

PROVIDER: PRJNA666932 | ENA |

REPOSITORIES: ENA

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Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies

Project description:A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging. We have developed two novel methods for constructing a nanobody library in mammalian cells. Complementarity-determining region (CDR) random sequences were first incorporated into upstream and downstream dsDNAs by PCR. In the first method, named dsDNA-HR, upstream and downstream dsDNAs containing an identical overlapping sequence were co-transfected into cultured mammalian cells for intracellular homologous recombination that resulted in the formation of an intact nanobody library expression cassette. In the second method, named in vitro ligation, we generated full-length nanobody expression dsDNAs via ligation of restriction digested upstream and downstream dsDNAs. The obtained full-length dsDNAs were transfected into mammalian cells for nanobody library expression. Using both methods, we generated over a million unique nanobody sequences, as revealed by high-throughput sequencing. Single-cell sequencing was employed to resolve the diversity of the dsDNA-HR nanobody library. We also identified a small molecule, Nocodazole, which could enhance the efficacy of dsDNA-HR.

2021-09-21 | GSE158906 | GEO

Construction of Nanobody Library in Mammalian Cells by Linear-double-stranded DNA Based AND Gate Genetic Circuit

Project description:Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate genetic circuits in mammalian cells. During this AND-gate circuit formation process, the co-transfected up- and down-stream ldsDNAs could be linked together to form intact gene expression cassette. Here, we employed this ldsDNA-based AND-gate (LBAG) strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down- stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library, which is a start point for building high content nanobody library in mammalian cells.

2021-07-24 | GSE155159 | GEO

piggyBac-Mediated Genomic Integration of Linear dsDNA-Based Library for Deep Mutational Scanning in Mammalian Cells

Project description:Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here we developed two novel strategies for DMS library construction in mammalian cells, namely ‘piggyBac-in-vitro ligation’ and ‘piggyBac-in-vitro ligation-PCR’. For the first strategy, we took the ‘in-vitro ligation’ approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in-vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino acid sequences. And we further identified a novel transcriptional activator peptide with the ‘piggyBac-in-vitro ligation-PCR’ strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.

2023-10-05 | GSE226719 | GEO

Construction of Nanobody Library in Mammalian Cells by Linear-double-stranded DNA Based AND Gate Genetic Circuit

Project description:Construction of Nanobody Library in Mammalian Cells by Linear-double-stranded DNA Based AND Gate Genetic Circuit

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unclassified dsDNA viruses

Project description:unclassified dsDNA viruses

| PRJNA16958 | ENA

unclassified dsDNA phages

Project description:unclassified dsDNA phages

| PRJNA16964 | ENA

Biparatopic anti-spike nanobody

Project description:Resistance to anti-spike nanobody.

| PRJEB49553 | ENA

unclassified dsDNA viruses

Project description:unclassified dsDNA viruses Metagenomic assembly

| PRJNA588800 | ENA

Expression data from dsDNA-stimulated mouse embryonic fibroblasts

Project description:Transfection of dsDNA into many mammalian cell types indues the production of type I interferons and interferon-stimulated genes. We performed an siRNA screen to identify genes involved in this innate immune response, and identified Abcf1. We used microarrays to determine which genes are regulated by ABCF1 following dsDNA stimulation. We treated p53-/- mouse embryonic fibroblasts (MEFs) with siRNAs targeting Abcf1 or Irf3 (positive control) or a negative control siRNA (siCtrl), and then stimulated the cells with 45-base pair dsDNA for 6 hrs or left the cells unstimulated. We then lysed the cells and hybridized the RNA to Affymetrix microarrays.

2012-12-08 | E-GEOD-42803 | biostudies-arrayexpress

RNA-seq comparison: before/after MARIS protocol and library construction technique test

Project description:The aim of this experiment was to determine the amount of RNA-seq signal degradation that results from MARIS and to test how well 4 different RNA-seq library construction techniques perform on partially degraded RNA.

2015-03-19 | E-MTAB-3424 | biostudies-arrayexpress

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