cu-cpt9a  (InvivoGen)

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: The assay system for human genome correction through intra-cellular circular donor cleavage-mediated targeted duplication and natural replacement. (A) The hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) gene is located between the PHF6 gene and the RPL36AP54 microRNA on the X chromosome in humans. It consists of nine exons and eight introns. The 5.4-kb section spanning intron 1, exon 2, intron 2, exon 3, and intron 3 was used as the targeting DNA, which is a probe used to search for homology across the entire chromosome. This 5.4-kb DNA is cloned into the pMB1KmHygTK plasmid vector ( Materials and Methods ), which has the bacterial replication origin (pMB1), the bacterial kanamycin-resistance gene ( Km ), and the fusion gene of the mammalian Hyg (hygromycin-resistant) gene with the HSV1tk (ganciclovir-sensitive) gene ( HygTK ). The I-SceI meganuclease-recognition site ISCEI and the designed sequence Syn are introduced into intron 1 and exon 2 of the cloned 5.4-kb DNA, respectively ( Materials and Methods ). SEXA1 , labeled on exon 2 of the targeted locus, indicates the wild-type allele ( SEXA1 allele), which is replaced with the Syn sequence ( Syn allele) in this assay system. The circular donor plasmid DNA consists of vector DNA (pink line) and homologous DNA (blue line). (B) A Syn -designed donor plasmid has synonymous codons (green letters) close to the SEXA1 site and is transfected to human cells with an artificial intra-cellular cleaver gene nls-I-SceI expression vector. The ISCEI sequence (red letters) of the Syn -designed donor plasmid is cleaved by the nls-I-SceI endonuclease. (C) Targeted duplication (TD) pathway: Homologous recombination between a Syn -designed donor plasmid and its homologous region around the exon 2 (TD1) produces a targeted duplication clone (TD2), which has a duplication structure with the Syn sequence and the SEXA1 sequence ( Syn / SEXA1 allele). The targeted duplication structure is naturally resolved through DNA replication during the S phase to form a single structure containing either the Syn allele (bold in TD3-1) or the SEXA1 allele (bold in TD3-2). Random integration (RI) pathway: Non-homologous recombination between the Syn -designed donor plasmid and a random site (RI1) produces a random integration clone (RI2). (D) The circular Syn -designed donor plasmid accesses the targeted counter chromosomal region (upper) to form the Syn -retaining targeted duplication structure, Syn / SEXA1 (center), which is confirmed by a couple of PCR-based tests as follows: (1) the duplication test is the PCR with the target-5′-outside forward primer (closed red arrow) and the plasmid-3′-inside reverse primer (closed violet arrow) and the other PCR with the plasmid-5′-inside forward primer (open violet arrow) and the target-3′-outside reverse primer (open red arrow); (2) the allele sequence test is the PCR with the Ex2–5′-outside primer and the Ex2–3′-outside primer (closed orange arrows), followed by its sequencing to verify the doublet chart of Syn and SEXA1 allele sequences (lower). (E) The natural replacement structure containing the Syn allele or the SEXA1 allele (center) is formed from a typical-targeted duplication structure (upper) by the popping-out of the circular DNA, which is confirmed using the following three tests: (1) the pop-out test is the PCR using target-5′-outside forward primer 2 (closed blue arrow) and target-3′-outside reverse primer 2 (open blue arrow) to verify the size characteristic of a natural replacement structure (the original size); (2) the Hyg test is the PCR with the 5′- Hyg forward primer and the 3′- Hyg reverse primer (closed green arrows) to verify the absence of the Hyg gene because the circular DNA that has popped out is removed by degradation or segregation in natural replacement cells; (3) the allele sequence test is the PCR with the Ex2–5′-outside forward primer and the Ex2–3′-outside reverse primer (closed orange arrows), followed by its sequencing to determine the sequence of the allele of a replacement clone, as shown in either a singlet chart of the Syn or SEXA1 allele sequence (lower).

Article Snippet: Ganciclovir (1 μM; InvivoGen) or HAT supplement (50×) (Gibco) was used for the selection of natural replacement clones derived from a targeted duplication clone.

Techniques: Clone Assay, Plasmid Preparation, Sequencing, Labeling, Transfection, Expressing, Homologous Recombination

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Demonstration for naturally generated replacement of the target sequence with the designed sequence. (A) Syn -retaining targeted duplication ( Syn -TD) clone HTG786 (5′ SEXA1 / Syn 3′) or a Syn -TD well clone d9p5F12 is shown with a hetero-allele genotype in the first column. The total cells (third column) were plated onto the dishes (second column). The number of viable cells plated and the density of the viable cells (fourth column) are shown. GCV R /HAT R (ganciclovir-resistant clones/hypoxanthine–aminopterin–thymidine-resistant ( HPRT + ) clones; last column) is shown as the rate of the GCV R frequency (fifth column) relative to that of the HAT R frequency (sixth column). The viable cells plated were calculated by multiplying the number of total cells plated by CFU (colony forming units)/500 total cells. The density of viable cells was calculated by dividing the number of viable cells plated by the area (cm 2 ) of the culture ware used. The GCV R frequency was calculated by dividing the number of all GCV R colonies generated by the viable cells plated. The HAT R frequency was calculated by dividing the total number of HAT R colonies generated or the total number of HAT R colonies calculated from HAT R colonies generated on two dishes by the number of viable cells plated. Adv: Advanced DMEM-based culture medium ( Materials and Methods ) was used to improve viability of HT1080-derived cells on culture ware. (B) Four GCV R clones (GCV-A1–A4) derived from HTG786, two GCV R clones (GCV-B1–B2) derived from HTG786, five GCV R clones (GCV-C1–C5) derived from d9p5F12 (A) , and nine GCV R clones (GCV-D1–D9) derived from d9p5F12 (A) were analyzed using three PCR-based assays as follows: (1) pop-out test ; (2) Hyg test ; and (3) duplication test , with PCR products from each assay electrophoresed (the duplication test was performed to verify whether the duplication structure was retained). HTG786 is a TD clone with 5′ SEXA1 / Syn 3’. HTG1047 is a TD clone with 5′ Syn / SEXA1 3’. (C) A TD structure with SEXA1 / Syn hetero-allele sequences forms one of the two types of natural replacement structures: with the Syn allele (left) or with the SEXA1 allele (right), which was verified by three PCR-based assays as follows: (1) pop-out test (closed blue arrow and open blue arrow) to verify the size characteristic of a natural replacement structure; (2) Hyg test (green arrows) to verify the absence of the Hyg gene because of the popping out of the circular plasmid DNA; and (3) allele sequence test (orange arrows) to determine the sequence of the allele of a replacement clone. One TD clone HTG786 is shown in the first column. In the following five columns, the numbers show the obtained GCV R or HAT R colonies, the Syn allele clones, the SEXA1 allele clones, the TK-deficient TD clones, in which duplication tests were still positive in panel (B) , and the others (TD-derived deletion clones, in which all of pop-out test, Hyg test, and duplication test were negative in panel (B) ). The complete results of the Syn allele or the SEXA1 allele of pop-out-type GCV or HAT clones are summarized in . The complete results of the HygTK sequence in TK-deficient TD-type GCV clones are summarized in .

Article Snippet: Ganciclovir (1 μM; InvivoGen) or HAT supplement (50×) (Gibco) was used for the selection of natural replacement clones derived from a targeted duplication clone.

Techniques: Generated, Sequencing, Clone Assay, Derivative Assay, Plasmid Preparation

Journal: Advanced Science

Article Title: Logic‐Gated HSV‐TK/GCV Suicide Gene Circuit for Triple‐Negative Breast Cancer

doi: 10.1002/advs.202514749

Figure Lengend Snippet: The BRAS circuit for breast cancer‐specific expression of therapeutic genes HSV‐TK in breast cancer cell lines and primary breast cancer cells. (A) Genetic configuration of the BRAS circuit encoding the therapeutic HSV‐TK gene and miR205. (B) Experimental timeline: breast cancer cells (3 × 10 4 cells/well) were plated in 48‐well plates for 24 h, then cotransduced with lentiviral vectors encoding the BRAS circuit and HSV‐TK/mi205 output. GCV (3 mg/mL) was added 24 h post‐transduction, and cytotoxicity was evaluated after 48 h of GCV exposure. (C,D) Cell viability was measured in BRAS‐transduced MDA‐MB‐231, MDA‐MB‐453, and BT549 breast cancer lines and normal MCF10A cells after 72 h using the CCK‐8 assay (C) and LDH release assay (D). (E) Annexin V‐FITC/PI staining and flow cytometry analysis of apoptosis in BRAS‐transduced cells at 72 h. (F) Workflow for isolating primary TNBC patient‐derived cancer cells. (G,H) Viability assessment of patient‐derived TNBC cells and normal epithelial cells after BRAS‐mediated HSV‐TK/GCV treatment using CCK‐8 (G) and LDH release assays (H). Cells were cotransduced with the lentiviral vectors encoding the BRAS circuit and HSV‐TK/mi205 and treated with 3 mg/mL GCV for 72 h. Data in (C–E,G,H) are expressed as mean ± SD ( n = 3 independent experiments). Statistical comparisons were made using one‐way ANOVA with multiple comparisons or two‐tailed unpaired t ‐tests. **** P < 0.0001.

Article Snippet: Cells were transduced with lentiviral vector encoding the BRAS circuit: pST168 (LTR‐P RRM2 ‐Coh2‐linker‐p65‐HSF1‐interval‐P MAFK ‐Gal4‐linker‐DocS‐LTR‐spacer‐LTR‐5×UAS‐P hCMVmin ‐HSV‐TK‐miR205‐LTR, MOI = 3) in combination with GCV (Catalog no. sud‐gcv, InvivoGen) in the presence of 10 μg/mL polybrene (Catalog no. H9268; Sigma‐Aldrich) for 48 h. 10 μL of CCK‐8 solution was added to each well and incubated for 1 h at 37°C.

Techniques: Expressing, Transduction, CCK-8 Assay, Lactate Dehydrogenase Assay, Staining, Flow Cytometry, Derivative Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Logic‐Gated HSV‐TK/GCV Suicide Gene Circuit for Triple‐Negative Breast Cancer

doi: 10.1002/advs.202514749

Figure Lengend Snippet: In vivo efficacy of the BRAS circuit‐mediated gene therapy prototype in an orthotopic TNBC mouse model. (A) Schematic illustration of the experimental timeline and procedure for evaluating the therapeutic efficacy of the BRAS circuit in an orthotopic BT549 TNBC mouse model. Female BALB/c nude mice (6 weeks old) were orthotopically injected with BT549 cells (2 × 10 6 cells/mouse) into the mammary fat pad. Once tumors reached 30–50 mm 3 , mice were randomly assigned to four groups: (G1) PBS control, (G2) lentiviral vector encoding HSV‐TK, (G3) GCV alone, and (G4) lentiviral vector encoding BRAS circuit and HSV‐TK/miR205 output. Intratumoral injections of PBS, lentiviral vector encoding HSV‐TK/miRNA, or lentiviral vector encoding BRAS circuit and HSV‐TK/miR205 were administered on days 0, 4, 8, 12, and 16. Groups G3 and G4 also received daily intraperitoneal injections of GCV (50 mg/kg) from day 2 to day 20. (B) Tumor volumes were measured every four days until day 28 ( n = 5). (C) Body weight was monitored throughout the experiment for all groups. (D,E) Representative images of excised tumors (D) and corresponding tumor weights (E). (F) Histological and immunohistochemical analyses of tumor sections collected at endpoint. Tumors were stained for Ki67 (brown) to assess proliferation and for TUNEL (green) to detect apoptosis, with DAPI (blue) counterstaining nuclei. Scale bar = 50 µm. Data in (B,C,E) are presented as mean ± SEM ( n = 5 mice/group). Statistical significance was evaluated using two‐way ANOVA followed by Sidak's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were transduced with lentiviral vector encoding the BRAS circuit: pST168 (LTR‐P RRM2 ‐Coh2‐linker‐p65‐HSF1‐interval‐P MAFK ‐Gal4‐linker‐DocS‐LTR‐spacer‐LTR‐5×UAS‐P hCMVmin ‐HSV‐TK‐miR205‐LTR, MOI = 3) in combination with GCV (Catalog no. sud‐gcv, InvivoGen) in the presence of 10 μg/mL polybrene (Catalog no. H9268; Sigma‐Aldrich) for 48 h. 10 μL of CCK‐8 solution was added to each well and incubated for 1 h at 37°C.

Techniques: In Vivo, Drug discovery, Injection, Control, Plasmid Preparation, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Advanced Science

Article Title: Logic‐Gated HSV‐TK/GCV Suicide Gene Circuit for Triple‐Negative Breast Cancer

doi: 10.1002/advs.202514749

Figure Lengend Snippet: In vivo efficacy of the BRAS circuit‐mediated gene therapy prototype in immunocompetent Balb/c mice bearing EMT6 cells. (A) Schematic illustration of the experimental timeline and procedure for evaluating the therapeutic efficacy of the BRAS circuit in an orthotopic EMT6 mouse model. Female immunocompetent Balb/c mice (6–8 weeks old) were orthotopically injected with EMT6 cells (1 × 10 6 cells/mouse) into the mammary fat pad. Once tumors reached 100–150 mm 3 , mice were randomly assigned to four groups: G1, phosphate‐buffered saline (PBS); G2, BRAS circuit vector with HSV‐TK output alone; G3, GCV; G4, BRAS circuit vector with HSV‐TK/miR205 output plus GCV. Intratumoral injections of PBS, lentiviral vector encoding HSV‐TK/miRNA, or lentiviral vector encoding BRAS circuit and HSV‐TK/miR205 were administered on days 0, 4, 8, 12, and 16. Groups G3 and G4 also received daily intraperitoneal injections of GCV (50 mg/kg) from day 2 to day 20. (B) Tumor volumes were measured every four days until day 24 ( n = 4). (C) Body weight was monitored throughout the experiment for all groups. (D,E) Representative images of tumors (D) and corresponding tumor weights (E). (F) Representative images of HE, Ki67 (brown), TUNEL (green), CD4 + (brown), and CD8 + (brown) T cells staining in the tumors of different treatment groups. DAPI (blue) was stained for nuclei. Scale bar = 50 µm. (G) Representative hematoxylin and eosin (H&E) staining images of major organs (lung, liver, spleen, kidney and heart) from BALB/c immunocompetent mice after 24 days of treatment. Scale bar = 100 µm. Data in (B,C,E) are presented as mean ± SEM ( n = 4 mice/group). Statistical significance was evaluated using two‐way ANOVA followed by Sidak's multiple comparisons test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were transduced with lentiviral vector encoding the BRAS circuit: pST168 (LTR‐P RRM2 ‐Coh2‐linker‐p65‐HSF1‐interval‐P MAFK ‐Gal4‐linker‐DocS‐LTR‐spacer‐LTR‐5×UAS‐P hCMVmin ‐HSV‐TK‐miR205‐LTR, MOI = 3) in combination with GCV (Catalog no. sud‐gcv, InvivoGen) in the presence of 10 μg/mL polybrene (Catalog no. H9268; Sigma‐Aldrich) for 48 h. 10 μL of CCK‐8 solution was added to each well and incubated for 1 h at 37°C.

Techniques: In Vivo, Drug discovery, Injection, Saline, Plasmid Preparation, TUNEL Assay, Staining