Monoclonal antibody profiling of cell surface proteins associated with the viral biofilms on HTLV-1 transformed cells

Tarasevich, A; Filatov, Alexander; Пичугин, А.В.; Mazurov, Dmitriy

doi:10.4149/av_2015_03_247

Cited by 23 publications

(18 citation statements)

References 24 publications

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“…The pHIV-1-GFPt vector encoding NL4-3 strain of HIV-1 with partial deletions in Env and Nef was derived from pHIG(ON) (a gift from Dr. W. S. Hu, NCI-Frederick) by subcloning gfp-turbo gene (Evrogen, Russia) into Nef region. The gRNA expressing vector pKS gRNA BB and the plasmid for the expression of wild type or nickase mutant of Cas9 (Addgene #41815) were described earlier 1,42 . All PCR DNA fragments prepared for cloning were generated using Pfu polymerase (Sibenzyme, Russia) and verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

Zotova

Пичугин

Atemasova

et al. 2019

Sci Rep

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We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.

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Section: Methodsmentioning

confidence: 99%

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

Zotova

Пичугин

Atemasova

et al. 2019

Sci Rep

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“…The plasmid pcDNA3.3-Cas9n, encoding Cas9 nickase (Cas9n), was derived from the pcDNA3.3-Cas9 plasmid (Addgene #41815) [ 28 ] by introducing a D(10)A mutation into Cas9 coding sequence. The pKS-gRNA-BB vector, designed for custom sgRNA expression, was generated as described [ 29 ]. pKS-gRNA-BB contains a U6 RNA polymerase III promoter required for sgRNA transcription, two BbsI restriction sites for cloning of a synthetic double-stranded oligodeoxynucleotide (dsODN) encoding the gene-specific part of sgRNA, and a sequence encoding the constant part of sgRNA [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

et al. 2016

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Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.

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“…Finally, the CMV promoter from the pCMV-Tet3G vector (Clontech, Mountain View, CA, USA) was subcloned into the donor vector at EcoRI/XhoI restriction sites. The sgRNA expressing vector pKS gRNA BB, the plasmids for the expression of wild-type Cas9 (Addgene #41815), and its nickase mutant D10A were described earlier [ 18 ]. The expression plasmids for the low off-target versions of Cas9 (pcDNA 3.3-eCas9 and pcDNA 3.3-eCas9n) were generated by introducing three mutations (K848A, K1003A, and R1060A [ 14 ]) into the Cas9 sequence.…”

Section: Methodsmentioning

confidence: 99%

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method

Zotova

Lopatukhina

Filatov³

et al. 2017

Viruses

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Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

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Monoclonal antibody profiling of cell surface proteins associated with the viral biofilms on HTLV-1 transformed cells

Cited by 23 publications

References 24 publications

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags

The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method

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