Transfection of lymphoblastoid cells using DNA-loaded reconstituted Sendai virus envelopes: Expression of transfected DNA and selection of transfected cells

1986 ◽  
Vol 12 (4) ◽  
pp. 351-356 ◽  
Author(s):  
I. M. Shapiro ◽  
Mario Stevenson ◽  
Faruk Sinangil ◽  
David J. Volsky
Keyword(s):  
Sendai Virus ◽  
Lymphoblastoid Cells ◽  
Transfected Cells ◽  
Selection Of

scholarly journals Enhancement of Hepatitis C Virus RNA Replication by Cell Culture-Adaptive Mutations

2001 ◽  
Vol 75 (10) ◽  
pp. 4614-4624 ◽  
Author(s):  
Nicole Krieger ◽  
Volker Lohmann ◽  
Ralf Bartenschlager
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Colony Formation ◽  
Marker Gene ◽  
Rna Replication ◽  
Clear Correlation ◽  
Transfected Cells ◽  
Adaptive Mutations ◽  
Selection Of

ABSTRACT Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

scholarly journals Magnetic Selection of Transiently Transfected Cells

BioTechniques ◽  
1996 ◽  
Vol 21 (5) ◽  
pp. 876-880 ◽  
Author(s):  
Stefan Schneider ◽  
Sandro Rusconi
Keyword(s):  
Transfected Cells ◽  
Selection Of

Magnetic Selection of Transfected Cells

2000 ◽  
pp. 210-217
Author(s):  
Gregor Siebenkotten ◽  
Ute Behrens-Jung
Keyword(s):  
Transfected Cells ◽  
Selection Of

Selection of Transfected Cells: Magnetic Affinity Cell Sorting

2003 ◽  
pp. 343-358
Author(s):  
Raji Padmanabhan ◽  
Snorri S. Thorgeirsson ◽  
R. Padmanabhan
Keyword(s):  
Cell Sorting ◽  
Transfected Cells ◽  
Selection Of

scholarly journals The Activity of Sendai Virus Genomic and Antigenomic Promoters Requires a Second Element Past the Leader Template Regions: a Motif (GNNNNN)3 Is Essential for Replication

1998 ◽  
Vol 72 (4) ◽  
pp. 3117-3128 ◽  
Author(s):  
Caroline Tapparel ◽  
Diane Maurice ◽  
Laurent Roux
Keyword(s):  
Sequence Comparison ◽  
Sendai Virus ◽  
Rna Replication ◽  
Negative Polarity ◽  
Relative Positioning ◽  
Promoter Regions ◽  
Complementary Sequence ◽  
Replication Activity ◽  
Repeated Motif ◽  
Selection Of

ABSTRACT The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of ∼15 kb, contains six transcription units flanked at the 3′ and 5′ ends by a short (∼ 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3′ end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3′-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5′-3′ positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)3 from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.

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