Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 2. Development and characterization of TRE-Lox KI cell lines targeting the endogenous CTSD gene of mouse embryonic fibroblasts (MEFs). (a) Overview of the genotyping strategy, including the relative position of different primers (blue arrows) and the predicted sizes of different PCR amplicons (black boxes) used to distinguish different possible outcomes of insertion of the TRE-Lox construct via Cas9-assisted homologous recombination. The main possibilities include (but are not limited to) the following: (1) the unmodified endogenous murine CTSD allele (top); (2) insertion of the TRE-Lox KI insert as designed (middle); and (3) nonhomologous DNA end joining (NHEJ) resulting (in this case) in the excision of the segment of DNA between gRNA1 and gRNA2 (bottom, see Supplementary Materials Figure S1). (b) Genotyping of a subset of clones obtained after selection of individual puromycin-resistant clonal lines. The two bands within clone 1C8 (referred to as TL1C8) were excised, sequenced, and confirmed to be amplified from one allele carrying the TRE-Lox KI insert (upper band) and another allele featuring NHEJ, which results in functional knock-out (KO) of CTSD (sequences provided in Supplementary Materials Figure S5a–c). (c) CatD proteolytic activity in wild-type MEFs, or TL1C8 cells transiently transfected with empty vector (yellow), GFP (green) or Flp recombinase (dark blue). Note the low level of CatD activity in TL1C8 cells, which is reversed by transfection with Flp recombinase, resulting in activity close to the expected value of 50% of wild-type MEFs (gray dotted line). (d) CatD activity in several clonal lines of TL1C8-Flp cells stably expressing rtTRKRAB incubated for 4 d in the absence or presence of Dox (100 ng/mL). Note how, in the absence of Dox (white columns), all tested clones harbor CatD activity that is very close to 50% of the levels within MEFs (gray dotted line), as expected, whereas in the presence of Dox (gray columns), CatD activity is greatly decreased. Data in (c,d) are mean ± SEM of 4 replicates. * p < 0.05; ns = nonsignificant.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: Construct, Homologous Recombination, Clone Assay, Selection, Functional Assay, Knock-Out, Activity Assay, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Incubation

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 4. Functional and genotypic characterization of Cre-mediated genetic deletion of CatD made possible by the TRE-Lox system. (a) CatD activity in TL1C8-Flp cells transiently transfected with either GFP only (CTL, dark blue) or GFP-tagged Cre recombinase (Cre, green). Note that these are pools of GFP-positive cells selected by cell sorting 2 d after transfection. Data are mean ± SEM, n = 4,

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: Functional Assay, Activity Assay, Transfection, FACS

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 1. Overview of the design and dual-functionality of the TRE-Lox system. (a) Overall structure of the 5′ end of the murine cathepsin D (CatD) gene (CTSD) and its promoter region (PCTSD, dark gray), indicating the relative position of the two gRNAs (black arrows) used for CRISPR/Cas9- assisted homologous recombination. Note the placement of the TATA box (TATA) very close to the main transcription start site (TSS) (right-angle arrow), the presence of the initiation codon (ATG, dashed white line) within Exon 1 (Ex 1, light gray), and the presence of a splice donor (SD) and splice acceptor (SA) flanking Intron 1 (black line). (b) Structure of the TRE-Lox knock-in (KI) insert, illustrating the relative positions of the two tet-operons (tetO2, green) and one LoxP site (LoxP, light blue) within the 5′ untranslated region (5′UTR) and, within Intron 1, a tetracycline response element (TRE) comprised of seven tetO repeats (tetO7) and the second LoxP site. The relative placement of the puromycin resistance cassette (Puror, purple) flanked by two FRT sites (FRT, dark blue), which is excisable by Flp recombinase, is depicted using a curly bracket. (c) Downregulation of CTSD via the action of rtTRKRAB acting on the TRE-Lox insert. In the presence of Dox (red triangles), rtTRKRAB binds to the tetO repeats within both the 5′UTR and Intron 1, triggering methylation of histones in a radius of 2–3 kb, thereby remodeling the chromatin and silencing the CTSD gene. (d) Genetic deletion of CTSD via the action of Cre recombinase on the TRE-Lox insert. The figure depicts the end result of Cre-mediated recombination of the TRE-Lox KI insert, which causes removal of the initiation codon, the first portion of the coding region of Exon 1 encoding the signal peptide of CatD, and the 5′ end of Intron 1.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: CRISPR, Homologous Recombination, Knock-In, Methylation

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 2. Development and characterization of TRE-Lox KI cell lines targeting the endogenous CTSD gene of mouse embryonic fibroblasts (MEFs). (a) Overview of the genotyping strategy, including the relative position of different primers (blue arrows) and the predicted sizes of different PCR amplicons (black boxes) used to distinguish different possible outcomes of insertion of the TRE-Lox construct via Cas9-assisted homologous recombination. The main possibilities include (but are not limited to) the following: (1) the unmodified endogenous murine CTSD allele (top); (2) insertion of the TRE-Lox KI insert as designed (middle); and (3) nonhomologous DNA end joining (NHEJ) resulting (in this case) in the excision of the segment of DNA between gRNA1 and gRNA2 (bottom, see Supplementary Materials Figure S1). (b) Genotyping of a subset of clones obtained after selection of individual puromycin-resistant clonal lines. The two bands within clone 1C8 (referred to as TL1C8) were excised, sequenced, and confirmed to be amplified from one allele carrying the TRE-Lox KI insert (upper band) and another allele featuring NHEJ, which results in functional knock-out (KO) of CTSD (sequences provided in Supplementary Materials Figure S5a–c). (c) CatD proteolytic activity in wild-type MEFs, or TL1C8 cells transiently transfected with empty vector (yellow), GFP (green) or Flp recombinase (dark blue). Note the low level of CatD activity in TL1C8 cells, which is reversed by transfection with Flp recombinase, resulting in activity close to the expected value of 50% of wild-type MEFs (gray dotted line). (d) CatD activity in several clonal lines of TL1C8-Flp cells stably expressing rtTRKRAB incubated for 4 d in the absence or presence of Dox (100 ng/mL). Note how, in the absence of Dox (white columns), all tested clones harbor CatD activity that is very close to 50% of the levels within MEFs (gray dotted line), as expected, whereas in the presence of Dox (gray columns), CatD activity is greatly decreased. Data in (c,d) are mean ± SEM of 4 replicates. * p < 0.05; ns = nonsignificant.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: Construct, Homologous Recombination, Clone Assay, Selection, Functional Assay, Knock-Out, Activity Assay, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Incubation

Journal: International journal of molecular sciences

Article Title: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Figure 4. Functional and genotypic characterization of Cre-mediated genetic deletion of CatD made possible by the TRE-Lox system. (a) CatD activity in TL1C8-Flp cells transiently transfected with either GFP only (CTL, dark blue) or GFP-tagged Cre recombinase (Cre, green). Note that these are pools of GFP-positive cells selected by cell sorting 2 d after transfection. Data are mean ± SEM, n = 4,

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [33]), and pools of Flpe (and GFP-only control)transfected cells were isolated by FACS.

Techniques: Functional Assay, Activity Assay, Transfection, FACS

Journal: International Journal of Molecular Sciences

Article Title: A Dual-Function “TRE-Lox” System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Development and characterization of TRE-Lox KI cell lines targeting the endogenous CTSD gene of mouse embryonic fibroblasts (MEFs). ( a ) Overview of the genotyping strategy, including the relative position of different primers (blue arrows) and the predicted sizes of different PCR amplicons (black boxes) used to distinguish different possible outcomes of insertion of the TRE-Lox construct via Cas9-assisted homologous recombination. The main possibilities include (but are not limited to) the following: (1) the unmodified endogenous murine CTSD allele (top); (2) insertion of the TRE-Lox KI insert as designed (middle); and (3) nonhomologous DNA end joining (NHEJ) resulting (in this case) in the excision of the segment of DNA between gRNA1 and gRNA2 (bottom, see ). ( b ) Genotyping of a subset of clones obtained after selection of individual puromycin-resistant clonal lines. The two bands within clone 1C8 (referred to as TL1C8) were excised, sequenced, and confirmed to be amplified from one allele carrying the TRE-Lox KI insert (upper band) and another allele featuring NHEJ, which results in functional knock-out (KO) of CTSD (sequences provided in ). ( c ) CatD proteolytic activity in wild-type MEFs, or TL1C8 cells transiently transfected with empty vector (yellow), GFP (green) or Flp recombinase (dark blue). Note the low level of CatD activity in TL1C8 cells, which is reversed by transfection with Flp recombinase, resulting in activity close to the expected value of 50% of wild-type MEFs (gray dotted line). ( d ) CatD activity in several clonal lines of TL1C8-Flp cells stably expressing rtTRKRAB incubated for 4 d in the absence or presence of Dox (100 ng/mL). Note how, in the absence of Dox (white columns), all tested clones harbor CatD activity that is very close to 50% of the levels within MEFs (gray dotted line), as expected, whereas in the presence of Dox (gray columns), CatD activity is greatly decreased. Data in ( c , d ) are mean ± SEM of 4 replicates. * p < 0.05; ns = nonsignificant.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [ ]), and pools of Flpe (and GFP-only control)-transfected cells were isolated by FACS.

Techniques: Construct, Homologous Recombination, Clone Assay, Selection, Amplification, Functional Assay, Knock-Out, Activity Assay, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Incubation

Journal: International Journal of Molecular Sciences

Article Title: A Dual-Function “TRE-Lox” System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D

doi: 10.3390/ijms24076745

Figure Lengend Snippet: Functional and genotypic characterization of Cre-mediated genetic deletion of CatD made possible by the TRE-Lox system. ( a ) CatD activity in TL1C8-Flp cells transiently transfected with either GFP only (CTL, dark blue) or GFP-tagged Cre recombinase (Cre, green). Note that these are pools of GFP-positive cells selected by cell sorting 2 d after transfection. Data are mean ± SEM, n = 4, * p < 0.01. ( b ) CatD activity in multiple stable clones treated previously with Cre recombinase. Data are mean ± SEM, n = 4, * p < 0.01. ( c ) PCR genotyping of TL1C8-Flp cells before and after transfection with Cre recombinase. ( d ) Overview of the genotyping strategy based on the predicted consequences of Cre-mediated recombination of the TRE-Lox construct, including the relative position of different primers (blue arrows) and the predicted sizes of different PCR amplicons (black boxes) used to distinguish different possible outcomes.

Article Snippet: To remove the FRT-flanked puromycin resistance cassette, TL1C8 cells were subsequently transfected with an enhanced form of Flp recombinase (Flpe) fused with GFP (pCAG-Flpe:GFP; Addgene plasmid #13788 [ ]), and pools of Flpe (and GFP-only control)-transfected cells were isolated by FACS.

Techniques: Functional Assay, Activity Assay, Transfection, FACS, Clone Assay, Construct