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A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms.

Friedrich M, Meier D, Schuster I, Nellen W - PLoS ONE (2015)

Bottom Line: A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene.One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression.The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

View Article: PubMed Central - PubMed

Affiliation: Abt. Genetik, FB 10, Universität Kassel, Kassel, Germany.

ABSTRACT

Characteristics of dirs-1 mediated knock-downs: We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. ADVANTAGES OF THE DIRS-1–RNAI SYSTEM: The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5'-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

No MeSH data available.


Reduced transcript and protein levels of knock-down targets.(A) Northern blot analysis for abpA, corA and sevA mRNA levels in the respective knock-down strains relative to the Ax2 wild type control. A mixture of radiolabelled probes outside the trigger region (P10, P15, P16; see Fig 3) was used for hybridization. Equal loading was verified by rRNA staining with ethidium bromide. All samples were run on the same gel. (B) Representative Western blot of AbpA, SevA and CorA protein expression in the Ax2 wild type and the relevant knock-down line (n = 2). CorA or SevA was used as loading control. Quantification of signal intensity relative to the respective control protein was performed using ImageJ. Ax2: n = 8. Ax2 ITRs: n = 8. Error bars: mean with SD. ***, p<0,001. (C) Comparison of trans silencing on the protein level with opposing A15Ps and ITRs for abpA and corA relative to Ax2 wild type. CorA or SevA were used as loading controls.
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pone.0131271.g006: Reduced transcript and protein levels of knock-down targets.(A) Northern blot analysis for abpA, corA and sevA mRNA levels in the respective knock-down strains relative to the Ax2 wild type control. A mixture of radiolabelled probes outside the trigger region (P10, P15, P16; see Fig 3) was used for hybridization. Equal loading was verified by rRNA staining with ethidium bromide. All samples were run on the same gel. (B) Representative Western blot of AbpA, SevA and CorA protein expression in the Ax2 wild type and the relevant knock-down line (n = 2). CorA or SevA was used as loading control. Quantification of signal intensity relative to the respective control protein was performed using ImageJ. Ax2: n = 8. Ax2 ITRs: n = 8. Error bars: mean with SD. ***, p<0,001. (C) Comparison of trans silencing on the protein level with opposing A15Ps and ITRs for abpA and corA relative to Ax2 wild type. CorA or SevA were used as loading controls.

Mentions: We then asked the question if the trigger derived siRNAs could silence the corresponding endogenous gene. We examined mRNA from abpA, sevA and corA since they were expressed at sufficiently high levels to be detected by Northern blots. The analysis revealed a strong reduction of transcript levels for these knock-down strains relative to the Ax2 wild type (Fig 6A) and simultaneously a reduction on the protein level (Fig 6B). Though we did not attempt to quantify the amounts of siRNAs, mRNAs and proteins, it was rather obvious that there was no strict correlation. All endogenes were partially silenced but with different efficiency.


A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms.

Friedrich M, Meier D, Schuster I, Nellen W - PLoS ONE (2015)

Reduced transcript and protein levels of knock-down targets.(A) Northern blot analysis for abpA, corA and sevA mRNA levels in the respective knock-down strains relative to the Ax2 wild type control. A mixture of radiolabelled probes outside the trigger region (P10, P15, P16; see Fig 3) was used for hybridization. Equal loading was verified by rRNA staining with ethidium bromide. All samples were run on the same gel. (B) Representative Western blot of AbpA, SevA and CorA protein expression in the Ax2 wild type and the relevant knock-down line (n = 2). CorA or SevA was used as loading control. Quantification of signal intensity relative to the respective control protein was performed using ImageJ. Ax2: n = 8. Ax2 ITRs: n = 8. Error bars: mean with SD. ***, p<0,001. (C) Comparison of trans silencing on the protein level with opposing A15Ps and ITRs for abpA and corA relative to Ax2 wild type. CorA or SevA were used as loading controls.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482531&req=5

pone.0131271.g006: Reduced transcript and protein levels of knock-down targets.(A) Northern blot analysis for abpA, corA and sevA mRNA levels in the respective knock-down strains relative to the Ax2 wild type control. A mixture of radiolabelled probes outside the trigger region (P10, P15, P16; see Fig 3) was used for hybridization. Equal loading was verified by rRNA staining with ethidium bromide. All samples were run on the same gel. (B) Representative Western blot of AbpA, SevA and CorA protein expression in the Ax2 wild type and the relevant knock-down line (n = 2). CorA or SevA was used as loading control. Quantification of signal intensity relative to the respective control protein was performed using ImageJ. Ax2: n = 8. Ax2 ITRs: n = 8. Error bars: mean with SD. ***, p<0,001. (C) Comparison of trans silencing on the protein level with opposing A15Ps and ITRs for abpA and corA relative to Ax2 wild type. CorA or SevA were used as loading controls.
Mentions: We then asked the question if the trigger derived siRNAs could silence the corresponding endogenous gene. We examined mRNA from abpA, sevA and corA since they were expressed at sufficiently high levels to be detected by Northern blots. The analysis revealed a strong reduction of transcript levels for these knock-down strains relative to the Ax2 wild type (Fig 6A) and simultaneously a reduction on the protein level (Fig 6B). Though we did not attempt to quantify the amounts of siRNAs, mRNAs and proteins, it was rather obvious that there was no strict correlation. All endogenes were partially silenced but with different efficiency.

Bottom Line: A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene.One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression.The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

View Article: PubMed Central - PubMed

Affiliation: Abt. Genetik, FB 10, Universität Kassel, Kassel, Germany.

ABSTRACT

Characteristics of dirs-1 mediated knock-downs: We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. ADVANTAGES OF THE DIRS-1–RNAI SYSTEM: The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5'-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

No MeSH data available.