The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice

Petitclerc, D.; Attal, J.; Théron, Marie-Claire; Béarzotti, M.; Bolifraud, Philippe; Kann, G.; Stinnakre, Marie‐Georges; Pointu, Hervé; Puissant, C.; Houdebine, Louis

doi:10.1016/0168-1656(95)00047-t

Cited by 56 publications

(30 citation statements)

References 16 publications

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“…The presence of an intron within transcripts has been shown to increase the expression of some genes significantly (Choi et al, 1991 ;Petitclerc et al, 1995 ;Yew et al, 1997) and we also have obtained evidence that expression of a number of proteins in Ad vectors can be greatly enhanced by placing an intron upstream of the protein coding sequences (L. Prevec & F. L. Graham, unpublished observations). Consequently, we attempted to produce replication-defective Ad vectors in which the expression of RG was driven by the strong MCMV promoter (Dorsch-Hasler et al, 1985 ;Addison et al, 1997) in combination with an intron sequence previously used in Ad vectors (Berkner & Sharp, 1985).…”

Section: Resultsmentioning

confidence: 58%

“…Previous reports have shown that the presence of an intron in a transcript increases the levels of protein expression, presumably by favouring processing and transport to the cytoplasm of the transcript (Choi et al, 1991 ;Petitclerc et al, 1995 ;Yew et al, 1997). Since the presence of the intron in our construct precluded efficient rescue into recombinant Ad vectors in 293 cells, we reasoned that increased levels of RG expression might be inhibitory to Ad replication processes.…”

Section: Discussionmentioning

confidence: 99%

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Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

Matthews¹,

Cummings²,

Evelegh³

et al. 1999

Journal of General Virology

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An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein. The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG. Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter-intron expression cassette expressed RG at much higher levels than vectors lacking the intron. These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells.

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Section: Resultsmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

Matthews¹,

Cummings²,

Evelegh³

et al. 1999

Journal of General Virology

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“…The exact role of the 5'UTR from SV40 is not known. In a previous study, we observed that this 5'UTR is highly structured and that it does not alter the secondary structure of the HTLV-1 'R' region when associated to it (Attal et al, unpublished results, [21]). …”

Section: Discussionmentioning

confidence: 80%

“…They were introduced in the plasmid pPoly III as described in our previous studies (Attal et al, unpublished results, [21]). …”

Section: Gene Constructionsmentioning

confidence: 99%

“…The plasmid pRSV nee was added to the plasmids of interest containing the WAP gene promoter to obtain stable clones. The mixed clones were cultured to hyperconfluency in the presence of fetal calf serum and EGF (10 ng/ml) and induced by insulin (5 Ixg/ml), dexamethasone (10 -~ M) and ovine prolactin (NIH-PS13) (5 gg/ml) for 2 days in the absence of EGF and serum as described previously (Attal et al, unpublished results, [21]). Culture medium were collected at the end of the induction and bGH was measured using a specific radioimmunoassay (Attal et al, unpublished results, [21]).…”

Section: Cell Transfection and Protein Measurementsmentioning

confidence: 99%

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The RU5 (‘R’) region from human leukaemia viruses (HTLV‐1) contains an internal ribosome entry site (IRES)‐like sequence

Attal¹,

Théron²,

Taboit³

et al. 1996

FEBS Letters

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RNA fragments containing the complete R region and the beginning of the U5 region ('R') from the human T cell leukaemia virus 1 (HTLV-1) stimulated the translation of the second eistrons in bicistronic mRNAs. The 5' untranslated region from SV40 early genes (SU) which was unable to stimulate translation of second cistrons amplified markedly the internal ribosome entry site (IRES) effect of the HTLV-1 'R' fragments. The 'R' regions from HTLV-1 have therefore properties similar to internal ribosome entry sites (IRES) originally found in picornavirus. The beginning of the U5 region from HTLV-1 contains a polypyrimidine sequence which is known to play an essential role in the IRES activity in picornavirus. The same experiments carried out using the 'R' region from bovine leukaemia virus (BLV) showed that this sequence has at most a weak IRES effect. One retroviruses HTLV-1 and perhaps others contain therefore an IRES activity. Interestingly, the combined SU 'R' sequence worked efficiently with different cistrons, different promoters and in all tested cell lines, whereas the poliovirus IRES was active in CHO cells but not in the mouse mammary cell line HCll. The SU 'R' sequence may therefore preferably be used to generate active bicistronic mRNAs.

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Animal Transgenesis and Cloning

Houdebine¹

2003

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The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice

Cited by 56 publications

References 16 publications

Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

The RU5 (‘R’) region from human leukaemia viruses (HTLV‐1) contains an internal ribosome entry site (IRES)‐like sequence

Animal Transgenesis and Cloning

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