An Expression Vector Encoding alacZReporter Gene Facilitates Identification of Stable, High-Producing CHO Cell Clones

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ∼70 to 150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here, we report the first complete knockout (KO) of viral ORF57 gene from all ∼100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination with multiple rounds of cell selection and single-cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ. We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from the KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single-cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out the ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells, where each cell contains only a few copies of the KSHV genome. The ORF57 KO genome was stable in iSLK/Bac16 cells, and, upon lytic induction, was partially rescued by ectopic ORF57 to express viral lytic gene ORF59 and produce infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and the Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high copy numbers. IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology can be applied to knock out the ORF57 gene from all ∼100 copies of the KSHV genome in primary effusion lymphoma (PEL) cells by coexpressing two guide RNAs (gRNAs) and Cas9 from a single expression vector in combination with single-cell cloning. The gene knockout efficiency in this system was evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology also mediated ORF57 inversion in situ in the targeted site of the KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57 knockout (KO) genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of the virus genome and to understand their contributions to viral genetics and the virus life cycle.

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Long-term suppression of telomerase expression in HeLa cell clones, transfected with an expression vector carrying siRNA targeting hTERT mRNA

International Journal of Oncology ◽

10.3892/ijo.29.1.279 ◽

2006 ◽

Cited By ~ 1

Author(s):

Kaisa Kurvinen ◽

Stina Syrjänen ◽

Bo Johansson

Keyword(s):

Hela Cell ◽

Expression Vector ◽

Htert Mrna ◽

Cell Clones

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Screening of CHO cell clones expressing histidine-tagged major S hepatitis B surface protein using a semi-quantitative PCR protocol

Journal of Virological Methods ◽

10.1016/j.jviromet.2003.08.007 ◽

2003 ◽

Vol 114 (2) ◽

pp. 171-174 ◽

Cited By ~ 3

Author(s):

Carlos Otávio Alves Vianna ◽

Sergio da Silva e Mouta Junior ◽

Gerson de Oliveira da Silva ◽

Marcos da Silva Freire ◽

Marcia Terezinha Baroni de Moraes

Keyword(s):

Hepatitis B ◽

Quantitative Pcr ◽

Surface Protein ◽

Cho Cell ◽

Cell Clones ◽

Semi Quantitative Pcr

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306 GENERATION OF HUMAN A20 GENE-TRANSGENIC PORCINE FETAL FIBROBLASTS FOR SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab306 ◽

2008 ◽

Vol 20 (1) ◽

pp. 233 ◽

Cited By ~ 1

Author(s):

M. Oropeza ◽

B. Petersen ◽

N. Hornen ◽

D. Herrmann ◽

H. Niemann

Keyword(s):

Endothelial Cells ◽

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Cell Activation ◽

Expression Vector ◽

Cell Clone ◽

Cell Clones ◽

Fetal Fibroblasts ◽

A20 Gene

The aim of this project was to produce transgenic pigs with improved features in xenotransplantation, by expressing the human A20 gene to modulate the acute vascular rejection (AVR) reaction ocurring after porcine-to-human xenotransplantation. The A20 gene was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells (Opipari AW et al. 1990 J. Biol. Chem. 25, 14 705–14 708). The gene is both anti-apoptotic and anti-inflammatory in endothelial cells (Ferran C 2006 Transplantation 82(1 Suppl.), S36–S40) and could thus prevent endothelial cell activation leading to AVR. The hA20-expression vector driven by the CAGGS hybrid promoter (chicken β-actin–rabbit β-globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFF) derived from German Landrace porcine fetal explant cultures. Transfection of 3 � 106 cells was accomplished at 450 V and 350 µF with 10 µg of plasmid DNA. Then, G418-resistant cell clones (800 µg mL–1) were screened by PCR with hA20-specific primers for hA20 integration. Eighty clones were A20-positive in PCR screening from 4 rounds of transfection. One cell clone was verified by DNA sequencing and subsequently used as donor cells in somatic cell nuclear transfer. One hundred sixty-nine embryos were transferred to 2 synchronized peripuberal German Landrace gilts, respectively. Ultrasound examination of recipient sows on Day 22 after embryo transfer confirmed established pregnancies in both recipients. One pregnancy was allowed to go to term and 7 healthy piglets were born, whereas the second pregnancy was terminated on Day 70 of pregnancy for detailed expression analysis of the 8 isolated fetuses. Results showed that the A20 vector can be integrated in PFF, and A20-transgenic PFF can be successfully used in somatic cell nuclear transfer to establish pregnancies. Further analysis will focus on the expression levels and patterns in A20-positive cell clones and the biological function in transgenic piglets. Functional assays will be conducted in vitro and in vivo. We thank Prof. Beyaert of Ghent University, Belgium for providing us with the expression vector pCAGGSEhA20.

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Glycan‐masking hemagglutinin antigens from stable CHO cell clones for H5N1 avian influenza vaccine development

Biotechnology and Bioengineering ◽

10.1002/bit.26810 ◽

2018 ◽

Vol 116 (3) ◽

pp. 598-609 ◽

Cited By ~ 1

Author(s):

Ting‐Hsuan Chen ◽

Wen‐Chun Liu ◽

Chia‐Ying Lin ◽

Chia‐Chyi Liu ◽

Jia‐Tsrong Jan ◽

...

Keyword(s):

Avian Influenza ◽

Influenza Vaccine ◽

Vaccine Development ◽

Cho Cell ◽

H5n1 Avian Influenza ◽

Cell Clones

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Partially transformed, anchorage-independent human diploid fibroblasts result from overexpression of the c-sis oncogene: mitogenic activity of an apparent monomeric platelet-derived growth factor 2 species

Molecular and Cellular Biology ◽

10.1128/mcb.8.5.2089-2096.1988 ◽

1988 ◽

Vol 8 (5) ◽

pp. 2089-2096

Author(s):

C W Stevens ◽

W H Brondyk ◽

J A Burgess ◽

T H Manoharan ◽

B G Häne ◽

...

Keyword(s):

Growth Factor ◽

Membrane Fraction ◽

Expression Vector ◽

Cell Transformation ◽

Mitogenic Activity ◽

Platelet Derived Growth Factor ◽

Diploid Fibroblasts ◽

Human Diploid Fibroblasts ◽

Cell Clones ◽

Clear Link

A human c-sis cDNA in an expression vector was introduced into human diploid fibroblasts by transfection or electroporation. Fibroblast clones showing an aberrant, densely packed colony morphology were isolated and found to overexpress a 3.6-kilobase sis mRNA species and associated immunoprecipitable platelet-derived growth factor (PDGF) 2 proteins. Parallel analyses in cell clones of sis mRNA expression and colony formation in agar indicated that, above a threshold, a linear, positive correlation existed between sis overexpression and acquired anchorage independence. The sis-overexpressing cells formed transient, regressing tumor nodules when injected into nude mice, consistent with the finite life span which they retained. Protein products generated from the transfected c-sis construct in two overexpressing clones were immunoprecipitated with anti-human PDGF antibodies. One clone contained an apparent PDGF dimer of 21 kilodaltons; the second clone contained only an apparent PDGF monomer of 12 kilodaltons, which was shown to account for all of the mitogenic activity present in the cells, essentially all of which was concentrated in the membrane fraction. The results demonstrate a clear link between sis overexpression and acquisition of a partially transformed, anchorage-independent phenotype, and when combined with previous observations of sis overexpression in human tumors, clearly implicate sis overexpression as a genetic mechanism which contributes to human cell transformation.

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