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Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Matheson NJ, Peden AA, Lehner PJ - PLoS ONE (2014)

Bottom Line: Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads.Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications.We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, United Kingdom.

ABSTRACT
Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

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Related in: MedlinePlus

Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells.Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.
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pone-0111437-g003: Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells.Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.

Mentions: Expression of SBP-ΔLNGFR from the PGK promoter was noted to vary markedly according to the activation state of transduced T cells (Figure 3A). PGK encodes the glycolytic enzyme phoshpoglycerokinase, and glycolysis is known to be highly regulated in T cells [34]. To optimise the system for selecting primary human lymphocytes, we therefore introduced the SFFV promoter to drive expression of SBP-ΔLNGFR either as a single cistron (pHRSIREN-S-SBP-ΔLNGFR-W) or co-translated with an exogenous gene of interest via a P2A “self-cleaving” peptide linker for bicistronic expression (pHRSIN-SE-P2A-SBP-ΔLNGFR-W). These modifications increased SBP-ΔLNGFR expression without compromising levels of the co-expressed gene or shRNA of interest (Figure 3B). Expression levels were critically dependent on both the WPRE and the promoter strategy used, with inferior results obtained using the EF1a promoter, ECMV IRES or dual SFFV promoter systems, or when the WPRE was absent or alternatively located. The SFFV promoter is known to provide high-level transgene expression in primary human haematopoietic cells [14] and 2A peptides have been shown to enable stoichiometric co-expression of multiple cistrons across different organisms and cell types [15], [35]. These small viral peptide sequences are co-translationally “cleaved” in a process known as “ribosomal skipping” in which formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide is “skipped” without interrupting translation of the downstream polypeptide [36]. To test the optimised vectors (Figure 3C), we transduced primary human CD4+ T cells using 4 different constructs (expressing 2 different shRNAs and 2 different exogenous genes). From a starting purity of 31.0%, the average purity of selected cells was 99.2% (Figure 3D). We have not observed any functional deficits in a wide range of downstream applications (including viability, expression of activation markers, and proliferation; Figure 3E), and the Antibody-Free Magnetic Cell Sorting procedure (from incubation with magnetic beads through release with biotin) may be readily completed (including multiple samples) in <1 hr.


Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Matheson NJ, Peden AA, Lehner PJ - PLoS ONE (2014)

Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells.Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216076&req=5

pone-0111437-g003: Optimised Antibody Free Magnetic Cell Sorting of primary human CD4+ T cells.Primary human CD4+ T cells were lentivirally transduced with pHRSIREN/β2 m-PGK-SBP-ΔLNGFR-W (encoding shRNA to β2 m and SBP-ΔLNGFR under the PGK promoter) and either rested for 2 weeks (pale blue) or re-stimulated with CD3/CD28 Dynabeads 3 days prior to analysis (dark blue). Cells were co-stained with anti-HLA-A2-PE and anti-LNGFR-APC, and expression levels of SBP-ΔLNGFR compared in HLA-A2-low cells (A). Transduction with pHRSIN-SE-PGK-SBP-ΔLNGFR-W was then compared with pHRSIN-SE-P2A-SBP-ΔLNGFR-W (encoding GFP-P2A-SBP-ΔLNGFR under the SFFV promoter; B). Transduced cells are GFP+/LNGFR-APC+ (dashed circles). Background staining of untransfected/unstransduced controls is shown (grey). Finally, primary human CD4+ T cells were transduced with the optimised pHRSIREN-S-SBP-ΔLNGFR-W and pHRSIN-SE-P2A-SBP-ΔLNGFR-W lentivectors (C) encoding 2 different shRNAs and 2 different exogenous genes. Following selection with Dynabeads Biotin Binder, purity was assessed by staining with anti-LNGFR-PE (D). Each datapoint represents % LNGFR+ for a different construct (shRNA or exogenous gene) and means and SEMs are shown. Viability and functional activity of selected (expressing a control shRNA) and mock (unselected) cells were compared (E). Viability was measured 4 days after selection, and cells either rested or re-stimulated with CD3/CD28 Dynabeads. Resting and re-stimulated cells were stained with CD69-APC (day 2) and enumerated using CytoCount beads (days 1–3). CD69 expression by resting (grey) versus re-stimulated mock (pale blue) and selected (pink) cells is shown. Fold-increases in viable cell numbers following re-stimulation (proliferation) were calculated using day 1 as a baseline. Experiments were conducted in triplicate and means and SEMs are shown. cPPT – central polypurine tract; RRE – Rev response element; * – packaging signal; LTR – long terminal repeat; WPRE – Woodchuck Hepatitis Virus post-transcriptional regulatory element.
Mentions: Expression of SBP-ΔLNGFR from the PGK promoter was noted to vary markedly according to the activation state of transduced T cells (Figure 3A). PGK encodes the glycolytic enzyme phoshpoglycerokinase, and glycolysis is known to be highly regulated in T cells [34]. To optimise the system for selecting primary human lymphocytes, we therefore introduced the SFFV promoter to drive expression of SBP-ΔLNGFR either as a single cistron (pHRSIREN-S-SBP-ΔLNGFR-W) or co-translated with an exogenous gene of interest via a P2A “self-cleaving” peptide linker for bicistronic expression (pHRSIN-SE-P2A-SBP-ΔLNGFR-W). These modifications increased SBP-ΔLNGFR expression without compromising levels of the co-expressed gene or shRNA of interest (Figure 3B). Expression levels were critically dependent on both the WPRE and the promoter strategy used, with inferior results obtained using the EF1a promoter, ECMV IRES or dual SFFV promoter systems, or when the WPRE was absent or alternatively located. The SFFV promoter is known to provide high-level transgene expression in primary human haematopoietic cells [14] and 2A peptides have been shown to enable stoichiometric co-expression of multiple cistrons across different organisms and cell types [15], [35]. These small viral peptide sequences are co-translationally “cleaved” in a process known as “ribosomal skipping” in which formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide is “skipped” without interrupting translation of the downstream polypeptide [36]. To test the optimised vectors (Figure 3C), we transduced primary human CD4+ T cells using 4 different constructs (expressing 2 different shRNAs and 2 different exogenous genes). From a starting purity of 31.0%, the average purity of selected cells was 99.2% (Figure 3D). We have not observed any functional deficits in a wide range of downstream applications (including viability, expression of activation markers, and proliferation; Figure 3E), and the Antibody-Free Magnetic Cell Sorting procedure (from incubation with magnetic beads through release with biotin) may be readily completed (including multiple samples) in <1 hr.

Bottom Line: Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads.Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications.We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge, United Kingdom.

ABSTRACT
Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

Show MeSH
Related in: MedlinePlus