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Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.


Instability of the aptamer cassette in transduced CEMx174 cells. Genomic DNA from transduced cells and plasmid DNA from the different vectors were PCR amplified with a forward primer from GFP (3'GFP-for) and a reverse primer from the untranslated region of the vector (3' UTR-rev). PCR reactions were separated by gel electrophoresis and stained with ethidium bromide. The expected size of the MMP-70.15 amplicon is 654 bps; the expected size for the other bands varies depending on the length of the aptamer. The DNA ladders are either the 100 bp DNA ladder or the 1 kb DNA ladder from NEB.
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Figure 3: Instability of the aptamer cassette in transduced CEMx174 cells. Genomic DNA from transduced cells and plasmid DNA from the different vectors were PCR amplified with a forward primer from GFP (3'GFP-for) and a reverse primer from the untranslated region of the vector (3' UTR-rev). PCR reactions were separated by gel electrophoresis and stained with ethidium bromide. The expected size of the MMP-70.15 amplicon is 654 bps; the expected size for the other bands varies depending on the length of the aptamer. The DNA ladders are either the 100 bp DNA ladder or the 1 kb DNA ladder from NEB.

Mentions: To explain the lack of inhibition we observed in our aptamer-transduced CEMx174 cells, we hypothesized that the retroviral vector was rearranged during shuttle packaging and transduction. Since we had sorted the transduced CEMx174 cells for expression of GFP, any structural rearrangements affecting the inhibitor cassette would need to be downstream of the GFP sequences. Therefore, we designed PCR primers to specifically amplify the aptamer inhibitor cassette in the 3' UTR of MMP-GFP (Figure 1a). PCR amplification of the four-retroviral plasmids generated the correct sized bands (Figure 3), excluding the possibility of a PCR artifact. However, PCR amplification of genomic DNA from all four populations of aptamer-transduced CEMx174 cells generated two bands on the agarose gel (Figure 3) – one full-length product and another truncated product approximately 100–150 bps smaller than the full-length product.


Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Instability of the aptamer cassette in transduced CEMx174 cells. Genomic DNA from transduced cells and plasmid DNA from the different vectors were PCR amplified with a forward primer from GFP (3'GFP-for) and a reverse primer from the untranslated region of the vector (3' UTR-rev). PCR reactions were separated by gel electrophoresis and stained with ethidium bromide. The expected size of the MMP-70.15 amplicon is 654 bps; the expected size for the other bands varies depending on the length of the aptamer. The DNA ladders are either the 100 bp DNA ladder or the 1 kb DNA ladder from NEB.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211285&req=5

Figure 3: Instability of the aptamer cassette in transduced CEMx174 cells. Genomic DNA from transduced cells and plasmid DNA from the different vectors were PCR amplified with a forward primer from GFP (3'GFP-for) and a reverse primer from the untranslated region of the vector (3' UTR-rev). PCR reactions were separated by gel electrophoresis and stained with ethidium bromide. The expected size of the MMP-70.15 amplicon is 654 bps; the expected size for the other bands varies depending on the length of the aptamer. The DNA ladders are either the 100 bp DNA ladder or the 1 kb DNA ladder from NEB.
Mentions: To explain the lack of inhibition we observed in our aptamer-transduced CEMx174 cells, we hypothesized that the retroviral vector was rearranged during shuttle packaging and transduction. Since we had sorted the transduced CEMx174 cells for expression of GFP, any structural rearrangements affecting the inhibitor cassette would need to be downstream of the GFP sequences. Therefore, we designed PCR primers to specifically amplify the aptamer inhibitor cassette in the 3' UTR of MMP-GFP (Figure 1a). PCR amplification of the four-retroviral plasmids generated the correct sized bands (Figure 3), excluding the possibility of a PCR artifact. However, PCR amplification of genomic DNA from all four populations of aptamer-transduced CEMx174 cells generated two bands on the agarose gel (Figure 3) – one full-length product and another truncated product approximately 100–150 bps smaller than the full-length product.

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.