scholarly journals A Versatile Vector System for the Fast Generation of Knock-in Cell Lines with CRISPR

2020 ◽  
Author(s):  
Oscar Perez-Leal ◽  
Jonathon Nixon-Abell ◽  
Carlos A. Barrero ◽  
John Gordon ◽  
Mario C. Rico
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Protein Function ◽  
Vector System ◽  
Live Cells ◽  
Modular Construction ◽  
New Era ◽  
Recombination System ◽  
Endogenous Genes ◽  
Endogenous Proteins

ABSTRACTUntil recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

scholarly journals Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

2014 ◽  
Vol 25 (22) ◽  
pp. 3610-3618 ◽  
Author(s):  
Robert Mahen ◽  
Birgit Koch ◽  
Malte Wachsmuth ◽  
Antonio Z. Politi ◽  
Alexis Perez-Gonzalez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Quantitative Imaging ◽  
Live Cells ◽  
Single Molecule Spectroscopy ◽  
Mitotic Kinase ◽  
Endogenous Proteins

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

scholarly journals Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

2002 ◽  
Vol 76 (4) ◽  
pp. 1904-1913 ◽  
Author(s):  
Chunping Qiao ◽  
Juan Li ◽  
Anna Skold ◽  
Xudong Zhang ◽  
Xiao Xiao
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cost Effective ◽  
Helper Virus ◽  
Vector System ◽  
Wild Type ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Packaging Cell ◽  
Producer Cell

ABSTRACT The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

scholarly journals Destabilizing RET in targeted treatment of thyroid cancers

2016 ◽  
Vol 5 (1) ◽  
pp. 10-19 ◽  
Author(s):  
M L Gild ◽  
M Bullock ◽  
C K Pon ◽  
B G Robinson ◽  
R J Clifton-Bligh
Keyword(s):  
Thyroid Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Live Cells ◽  
Prolonged Treatment ◽  
Functional Study ◽  
Hsp90 Inhibition ◽  
Western Blots ◽  
Radioiodine Uptake ◽  
Thyroid Cancers

Metastatic differentiated thyroid cancers (DTC) are resistant to traditional chemotherapy. Kinase inhibitors have shown promise in patients with progressive DTC, but dose-limiting toxicity is commonplace. HSP90 regulates protein degradation of several growth-mediating kinases such as RET, and we hypothesized that HSP90 inhibitor (AUY922) could inhibit RET-mediated medullary thyroid cancer (MTC) as well as papillary thyroid cancer (PTC) cell growth and also radioactive iodine uptake by PTC cells. Studies utilized MTC cell lines TT (C634W) and MZ-CRC-1 (M918T) and the PTC cell line TPC-1 (RET/PTC1). Cell viability was assessed with MTS assays and apoptosis by flow cytometry. Signaling target expression was determined by western blot and radioiodine uptake measured with a gamma counter. Prolonged treatment of both MTC cell lines with AUY922 simultaneously inhibited both MAPK and mTOR pathways and significantly induced apoptosis (58.7 and 78.7% reduction in MZ-CRC-1 and TT live cells respectively, following 1 μM AUY922; P<0.02). Similarly in the PTC cell line, growth and signaling targets were inhibited, and also a 2.84-fold increase in radioiodine uptake was observed following AUY922 administration (P=0.015). AUY922 demonstrates in vitro activity against MTC and PTC cell lines. We observed a potent dose-dependent increase in apoptosis in MTC cell lines following drug administration confirming its anti-tumorigenic effects. Western blots confirm inhibition of pro-survival proteins including AKT suggesting this as the mechanism of cell death. In a functional study, we observed an increase in radioiodine uptake in the PTC cell line following AUY922 treatment. We believe HSP90 inhibition could be a viable alternative for treatment of RET-driven chemo-resistant thyroid cancers.

scholarly journals Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression.

1990 ◽  
Vol 10 (9) ◽  
pp. 4650-4657 ◽  
Author(s):  
P M Choi ◽  
K M Tchou-Wong ◽  
I B Weinstein
Keyword(s):  
Colon Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Control Cell ◽  
Tumor Promoter ◽  
Vector System ◽  
Human Colon Cancer ◽  
Kinase C

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.

Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression

1990 ◽  
Vol 10 (9) ◽  
pp. 4650-4657
Author(s):  
P M Choi ◽  
K M Tchou-Wong ◽  
I B Weinstein
Keyword(s):  
Colon Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Control Cell ◽  
Tumor Promoter ◽  
Vector System ◽  
Human Colon Cancer ◽  
Kinase C

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.

scholarly journals Packaging Cell Lines for Simian Foamy Virus Type 1 Vectors

1999 ◽  
Vol 73 (5) ◽  
pp. 4498-4501 ◽  
Author(s):  
Min Wu ◽  
Ayalew Mergia
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Virus Type ◽  
Foamy Virus ◽  
Vector System ◽  
Simian Foamy Virus ◽  
Packaging Cell Line ◽  
Packaging Cell ◽  
Foamy Viruses

ABSTRACT Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451–3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.

scholarly journals Novel miRNA Sponges to Specifically Modulate Gene Expression in Colon Cancer Cells

2019 ◽  
Author(s):  
Alejandro Aguilera ◽  
Gloria Perazzoli ◽  
Laura Cabeza ◽  
Cristina Mesa ◽  
Francisco Quiñonero ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Vector System ◽  
Egfp Expression ◽  
Expression Levels ◽  
Selective Blockade ◽  
Novel Mirna ◽  
Mirna Sponges ◽  
Green Fluorescent

miRNA sponges allow the selective blockade of a complete family of associated miRNAs which induce posttranscriptional gene silencing in its target through binding to 3&acute;UTR mRNA. MiRNA-365 and miRNA-145 are down-regulated in colorectal cancer (CRC), but not in health tissues. Based on this, we constructed two vectors by inserting miRNA sponge (one for miRNA-365 and other for miRNA-145), and used EGFP (enhanced green fluorescent protein) as a 3&prime; UTR reporter gene to analyse the ability of each sponge to catch its respective miRNA. qPCR results corroborated that the expression levels of both miRNAs were lower in CRC cell lines than in normal colon cell line. Flow cytometry analysis revealed a decrease of the EGFP expression levels in the cell lines transfected with both sponges, being higher on the normal cell line while CRC cell lines presented a minimal decline. Also, this decrease was inversely proportional to the levels of expression of both miRNAs obtained by qPCR. These results were corroborated by fluorescence microscopy, showing a similar decrease fluorescence. We propose a new vector system to carry in a specific way the expression of genes to CRC cells without affecting healthy cells, preventing damage to healthy tissues.

scholarly journals Ultrafast and Selective Labeling of Endogenous Proteins Using Affinity-based Benzotriazole Chemistry

2021 ◽  
Author(s):  
Xiaoyi Xin ◽  
Yu Zhang ◽  
Massimiliano Gaetani ◽  
Susanna Lundström ◽  
Roman Zubarev ◽  
...  
Keyword(s):  
Protein Function ◽  
Target Identification ◽  
Reaction Rate Constant ◽  
Live Cells ◽  
Selective Labeling ◽  
Systems Chemistry ◽  
Covalent Inhibitors ◽  
Chemical Modification Of Proteins ◽  
Endogenous Proteins

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28-42 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

The nuclear 3,5,3'-triiodothyronine receptor in human leukaemic cell lines

1984 ◽  
Vol 105 (3) ◽  
pp. 429-432 ◽  
Author(s):  
Juan Bernal ◽  
Leif C. Andersson
Keyword(s):  
Growth Hormone ◽  
Cell Line ◽  
Cell Lines ◽  
Rat Liver ◽  
Association Constant ◽  
Erythroid Differentiation ◽  
Leukaemic Cell ◽  
Cells In Culture ◽  
Gh Cells ◽  
High Concentrations

Abstract. The 3,5,3'-triiodothyronine (T3) receptor has been studied in a series of continuously growing human leukaemic cell lines. High concentrations of receptor were found in the erythroblastoid cell line K-562. T3 was bound to the nuclei of these cells with an association constant of 3.4 × 109 m−1, and capacity 104 fmol/100 μg DNA, or 8700 molecules/nucleus. This capacity is comparable to that of rat liver or growth hormone producing cells (GH cells) in culture, and suggests that the K-562 cell line could be a useful model for the study of T3 action on erythroid differentiation.

SYNTHESIS AND CYTOTOXICITY OF POLYHYDROXYLATED CHOLESTEROL DERIVATIVES

2018 ◽  
Vol 56 (4) ◽  
pp. 467
Author(s):  
Thu Thuy Thi Tran ◽  
Ha Thi Dinh ◽  
Phương Lan Doan ◽  
Long Quoc Pham ◽  
Quang Dai Ngo
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Addition Compound ◽  
Hep G2 ◽  
Physical Methods ◽  
Hepatocellular Carcinoma Cell Line ◽  
Human Hepatocellular Carcinoma Cell ◽  
Hepatocellular Carcinoma Cell

Eight polyhydroxylated cholesterol derivatives (1-8) were prepared from cholesterol, using oxidative reagents as SeO2, OsO4/NMO, HCOOH/H2O2 and BH3/ H2O2. Their structures were elucidated by using physical methods including NMR 1D and 2D. These compounds were evaluated against two cancer cell lines (Hep-G2, T98). Compounds 2, 4 and 8 inhibits human hepatocellular carcinoma cell line (Hep-G2) with IC50 4.69, 4.98 and 2.89 µg/mL, respectively. In addition, compound 8 exhibited strong cytotoxicity against T98 cell line (glioblastoma) with IC50 = 2.28 μM.

Sign in / Sign up

Export Citation Format

Share Document