Chromosomal Integration of Transduced Recombinant Baculovirus DNA in Mammalian Cells

ABSTRACT Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.

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Correction of Genetic Defects in Mammalian Cells by the Input of Small Amounts of Foreign Genetic Material

Journal of Cell Science ◽

10.1242/jcs.13.3.841 ◽

1973 ◽

Vol 13 (3) ◽

pp. 841-861

Author(s):

YVONNE L. BOYD ◽

H. HARRIS

Keyword(s):

Thymidine Kinase ◽

Mammalian Cells ◽

Hybrid Cell ◽

Genetic Material ◽

Chinese Hamster ◽

Heat Sensitivity ◽

Chinese Hamster Cells ◽

Inosinic Acid ◽

Mouse Cells

Chinese hamster cells lacking inosinic acid pyrophosphorylase and mouse cells lacking thymidine kinase were fused with chick erythrocytes. The resultant heterokaryons were cultivated in a selective medium in which possession of these enzymes was essential for cell survival and growth. Clones of cells able to grow in this medium were isolated and studied. A detailed karyological analysis of these clones failed to reveal any chick chromosomes; nor could any chick-specific antigens be detected on the surface of the cells. Nonetheless, clones arising from the fusion of chick erythrocytes with Chinese hamster cells were shown to possess an inosinic acid pyrophosphorylase which had the electrophoretic characteristics of chick inosinic acid pyrophosphorylase. However, the clones arising from the fusion of the chick erythrocytes with the mouse cells had a thymidine kinase with the electrophoretic mobility and heat sensitivity of murine, not chick, thymidine kinase. Both types of hybrid cell have now been cultivated in vitro for 18 months without the loss of thymidine kinase or inosinic acid pyrophosphorylase activity.

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A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽

10.3390/pr7050291 ◽

2019 ◽

Vol 7 (5) ◽

pp. 291 ◽

Cited By ~ 1

Author(s):

Chih-Yu Wu ◽

Chao-Wei Huang ◽

Yu-Shin Nai ◽

Pei-Yu Chu ◽

Chung-Hsiung Wang ◽

...

Keyword(s):

Recombinant Proteins ◽

Posttranslational Modifications ◽

Mammalian Cells ◽

Insect Cells ◽

Fluorescent Protein ◽

Expression System ◽

Recombinant Baculovirus ◽

New Combination ◽

Vector System ◽

Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

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Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System

International Journal of Molecular Sciences ◽

10.3390/ijms19123767 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3767 ◽

Cited By ~ 4

Author(s):

Qian Wang ◽

Jian Fang ◽

Qihua Pan ◽

Yizhou Wang ◽

Ting Xue ◽

...

Keyword(s):

Mammalian Cells ◽

Fluorescent Protein ◽

Recombinant Baculovirus ◽

Transgenic Fish ◽

Early Gene ◽

Drug Selection ◽

Baculovirus System ◽

Fish Cells

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin–green fluorescent protein (Puro-GFP, pf) or puromycin–red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

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VLPs Derived from the CCMV Plant Virus Can Directly Transfect and Deliver Heterologous Genes for Translation into Mammalian Cells

BioMed Research International ◽

10.1155/2019/4630891 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 5

Author(s):

María V. Villagrana-Escareño ◽

Elizabeth Reynaga-Hernández ◽

Othir G. Galicia-Cruz ◽

Ana L. Durán-Meza ◽

Viridiana De la Cruz-González ◽

...

Keyword(s):

Nucleic Acid ◽

Mammalian Cells ◽

Plant Virus ◽

Fluorescent Protein ◽

Genetic Material ◽

Eukaryotic Cell ◽

Plant Viruses ◽

Virus Capsids ◽

First Time ◽

Drug Nanocarriers

Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.

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Characterization of hCINAP, a Novel Coilin-interacting Protein Encoded by a Transcript from the Transcription Factor TAFIID32 Locus

Journal of Biological Chemistry ◽

10.1074/jbc.m501982200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36429-36441 ◽

Cited By ~ 24

Author(s):

Niovi Santama ◽

Stephen C. Ogg ◽

Anna Malekkou ◽

Spyros E. Zographos ◽

Karsten Weis ◽

...

Keyword(s):

Transcription Factor ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Protein Complexes ◽

Essential Gene ◽

Single Copy ◽

Cajal Body ◽

Cajal Bodies ◽

Binding Motif

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.

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Functional Display of an α2 Integrin-Specific Motif (RKK) on the Surface of Baculovirus Particles

Technology in Cancer Research & Treatment ◽

10.1177/153303460500400411 ◽

2005 ◽

Vol 4 (4) ◽

pp. 437-445 ◽

Cited By ~ 9

Author(s):

Reetta Riikonen ◽

Heli Matilainen ◽

Nina Rajala ◽

Olli Pentikäinen ◽

Mark Johnson ◽

...

Keyword(s):

Envelope Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Recombinant Baculovirus ◽

Autographa Californica ◽

Wild Type ◽

Receptor Binding Site ◽

Mammalian Cell Lines ◽

Green Fluorescent

The use of baculovirus vectors shows promise as a tool for gene delivery into mammalian cells. These insect viruses have been shown to transduce a variety of mammalian cell lines, and gene transfer has also been demonstrated in vivo. In this study, we generated two recombinant baculovirus vectors displaying an integrin-specific motif, RKK, as a part of two different loops of the green fluorescent protein (GFP) fused with the major envelope protein gp64 of Autographa californica M nucleopolyhedrovirus. By enzyme linked immunosorbent assays, these viruses were shown to bind a peptide representing the receptor binding site of an α2 integrin, the α2I-domain. However, the interaction was not strong enough to overcome binding of wild type gp64 to the unknown cellular receptor(s) on the surface of α2 integrin-expressing cells (CHO-α2β1) or enhance the viral uptake. After treatment of these cells with phospholipase C, internalization of all viruses was blocked or decreased significantly. However, one of the RKK displaying viruses, AcGFP(K)gp64, was still able to internalize into CHO-α2β1 cells, although at a lower level as compared to non-treated cells. This may indicate the possible utilization of a PLC independent alternative route via, in this case, the α2β1 integrin.

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Evidence for a Menkes-like protein with a nuclear targeting sequence

Biochemical Journal ◽

10.1042/bj3500855 ◽

2000 ◽

Vol 350 (3) ◽

pp. 855-863 ◽

Cited By ~ 11

Author(s):

Manchi C. M. REDDY ◽

Sudeep MAJUMDAR ◽

Edward D. HARRIS

Keyword(s):

Cell Nucleus ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Menkes Disease ◽

Transcript Variant ◽

Nuclear Localization Sequence ◽

Nuclear Targeting ◽

Metal Binding Domain

Extracts from three human cell lines were found to contain abridged Menkes disease gene transcripts with novel insertion sequences. The transcript variant that is the focus of the present study codes for a 103-residue protein containing the first heavy-metal-binding domain (Hmb1) of ATP7A, the Cu-ATPase associated with Menkes disease. This transcript variant has a 45-bp nucleotide insert interposed between exons 1 and 2 of ATP7A that starts with a 5´ ATG that is in-frame with the downstream ATG translation start site of ATP7A. We report here that the 66-bp nucleotides positioned between the upstream and downstream ATG sites encode 22 amino acid residues whose primary structure in part meets the criteria for a nuclear-localization sequence (NLS). We have referred to the transcript as nuclear Menkes-like (NML) 45. A green fluorescent protein (GFP) construct with NML45 when transfected in Chinese hamster ovary cells localized to the cell nucleus. A similar construct without the 66-bp segment exhibited a random dispersed fluorescent pattern in the cytosol. GFP constructs encoding ATP7A exons likewise failed to direct GFP into the cell nucleus, suggesting the nuclear determinant is not in an internal domain of the protein. The data suggest that the 22-residue segment contains an NLS for an 11.2-kDa protein with one Cu-binding site that may function as a chaperone to transport Cu into the nucleus of mammalian cells.

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RAPD isolation of a Y chromosome specific ORF in a dioecious plant, Silene latifolia

Genome ◽

10.1139/g01-151 ◽

2002 ◽

Vol 45 (2) ◽

pp. 413-420 ◽

Cited By ~ 15

Author(s):

Shunsuke Nakao ◽

Sachihiro Matsunaga ◽

Atsushi Sakai ◽

Tsuneyoshi Kuroiwa ◽

Shigeyuki Kawano

Keyword(s):

Y Chromosome ◽

Random Amplified Polymorphic Dna ◽

Single Copy ◽

Silene Latifolia ◽

Inverse Pcr ◽

Pcr Analysis ◽

Dioecious Plant ◽

Reading Frame ◽

Y Chromosomes ◽

Specific Fragment

Silene latifolia is a dioecious plant and has heteromorphic sex chromosomes: the X and Y chromosomes. The Y chromosome is the largest, and its genetic control seems to be most strict among dioecious plants. To identify the putative sex-determination elements on the Y chromosome, random amplified polymorphic DNA (RAPD) analysis was used to screen for Y chromosome specific DNA fragments, and 31 clones were successfully produced. Genomic Southern hybridization and FISH (fluorescence in situ hybridization) analyses revealed that one of the clones, #2-2, is a Y chromosome specific fragment that has a single copy on the Y chromosome. Sequence tagged site (STS)-PCR analysis also succeeded in amplifying one fragment in males and no fragments in females. Cloning and sequencing of the #2-2 flanking region using inverse PCR revealed an open reading frame (ORF) corresponding to 285 amino acids in length (ORF285), but no expression of the ORF285 gene was identified. ORF285 may be a clue to the origin of dioecy.Key words: Y chromosome, RAPD, STS, FISH, Melandrium album.

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Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4685 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4685-4689 ◽

Cited By ~ 66

Author(s):

L T Vassilev ◽

W C Burhans ◽

M L DePamphilis

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Single Copy ◽

Simian Virus ◽

Metabolic Inhibitors ◽

Ovary Cells ◽

Cellular Origin

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

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Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7480-7491.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7480-7491 ◽

Cited By ~ 172

Author(s):

Isabelle Hautefort ◽

Maria José Proença ◽

Jay C. D. Hinton

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Single Copy ◽

Green Fluorescent Protein Gene ◽

Gene Fusions ◽

Bacterial Gene ◽

Green Fluorescent

ABSTRACT We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as β-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.

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