Limits...
Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells.

Klanert G, Jadhav V, Chanoumidou K, Grillari J, Borth N, Hackl M - Biotechnol J (2014)

Bottom Line: Here we show that the substitution of chimeric sequences with CHO-specific sequences for expression of miRNA clusters yields significantly higher expression levels of the mature miRNA in the case of miR-221/222 and miR-15b/16.Our data suggest that the Drosha/Dgcr8-mediated excision from primary transcripts is reduced for chimeric miRNA sequences compared to the endogenous sequence.See accompanying commentary by Baik and Lee DOI: 10.1002/biot.201300503.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Boku University Vienna, Austria; ACIB GmbH, Austrian Centre of Industrial Biotechnology, Graz, Austria.

Show MeSH
Schematic representation of endogenous and artificial constructs for over-expression of microRNA clusters. (A) Endogenous mir-221/222 was PCR amplified from CHO-K1 genome, using primers 70 nt up and downstream of the genomic location. Primers contained restriction sites, which were used for cloning the sequence into a pcDNA 6.2 expression vector containing emGFP. Artificial constructs of ∼60 nucleotides are composed of CHO-specific mature miRNA sequences (solid lines) as well as the flanking and loop sequences of mir-155 (dotted lines). Artificial mir-221 and mir-222 were synthesized individually and cloned into the pcDNA 6.2 vector using restriction sites as indicated by black arrows. (B) A schematic of the pcDNA 6.2 expression vector used in this study, with CMV-controlled emGFP expression and microRNA cloning site contained in the 3'UTR of emGFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4282078&req=5

fig01: Schematic representation of endogenous and artificial constructs for over-expression of microRNA clusters. (A) Endogenous mir-221/222 was PCR amplified from CHO-K1 genome, using primers 70 nt up and downstream of the genomic location. Primers contained restriction sites, which were used for cloning the sequence into a pcDNA 6.2 expression vector containing emGFP. Artificial constructs of ∼60 nucleotides are composed of CHO-specific mature miRNA sequences (solid lines) as well as the flanking and loop sequences of mir-155 (dotted lines). Artificial mir-221 and mir-222 were synthesized individually and cloned into the pcDNA 6.2 vector using restriction sites as indicated by black arrows. (B) A schematic of the pcDNA 6.2 expression vector used in this study, with CMV-controlled emGFP expression and microRNA cloning site contained in the 3'UTR of emGFP.

Mentions: The chimeric miR-15b/16-2 and miR-221/222 clusters were created by concatenation of miRNA expression plasmids with artificial miRNA constructs (Fig. 1) as previously described [26, 32]. In short, the chimeric miRNAs, consisting of the mature CHO miRNA sequences with restriction sites on either end, and an optimized murine loop sequence (Supporting information, Table 1), were cloned into the 3' untranslated region (3'UTR) of emerald green fluorescent protein (emGFP) located in the pcDNA6.2–GW/EmGFP–miR vector (BLOCK–i™ Pol II miR RNAi Expression Vector Kit, Invitrogen Inc., Carlsbad, CA, USA), already containing artificial flanking regions. One of the two corresponding chimeric cluster miRNAs was cut out, including the artificial flanking regions, and inserted into the plasmid with the other chimeric cluster miRNA for artificial cluster generation (Fig. 1) according to the manufacturer's instructions.


Endogenous microRNA clusters outperform chimeric sequence clusters in Chinese hamster ovary cells.

Klanert G, Jadhav V, Chanoumidou K, Grillari J, Borth N, Hackl M - Biotechnol J (2014)

Schematic representation of endogenous and artificial constructs for over-expression of microRNA clusters. (A) Endogenous mir-221/222 was PCR amplified from CHO-K1 genome, using primers 70 nt up and downstream of the genomic location. Primers contained restriction sites, which were used for cloning the sequence into a pcDNA 6.2 expression vector containing emGFP. Artificial constructs of ∼60 nucleotides are composed of CHO-specific mature miRNA sequences (solid lines) as well as the flanking and loop sequences of mir-155 (dotted lines). Artificial mir-221 and mir-222 were synthesized individually and cloned into the pcDNA 6.2 vector using restriction sites as indicated by black arrows. (B) A schematic of the pcDNA 6.2 expression vector used in this study, with CMV-controlled emGFP expression and microRNA cloning site contained in the 3'UTR of emGFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282078&req=5

fig01: Schematic representation of endogenous and artificial constructs for over-expression of microRNA clusters. (A) Endogenous mir-221/222 was PCR amplified from CHO-K1 genome, using primers 70 nt up and downstream of the genomic location. Primers contained restriction sites, which were used for cloning the sequence into a pcDNA 6.2 expression vector containing emGFP. Artificial constructs of ∼60 nucleotides are composed of CHO-specific mature miRNA sequences (solid lines) as well as the flanking and loop sequences of mir-155 (dotted lines). Artificial mir-221 and mir-222 were synthesized individually and cloned into the pcDNA 6.2 vector using restriction sites as indicated by black arrows. (B) A schematic of the pcDNA 6.2 expression vector used in this study, with CMV-controlled emGFP expression and microRNA cloning site contained in the 3'UTR of emGFP.
Mentions: The chimeric miR-15b/16-2 and miR-221/222 clusters were created by concatenation of miRNA expression plasmids with artificial miRNA constructs (Fig. 1) as previously described [26, 32]. In short, the chimeric miRNAs, consisting of the mature CHO miRNA sequences with restriction sites on either end, and an optimized murine loop sequence (Supporting information, Table 1), were cloned into the 3' untranslated region (3'UTR) of emerald green fluorescent protein (emGFP) located in the pcDNA6.2–GW/EmGFP–miR vector (BLOCK–i™ Pol II miR RNAi Expression Vector Kit, Invitrogen Inc., Carlsbad, CA, USA), already containing artificial flanking regions. One of the two corresponding chimeric cluster miRNAs was cut out, including the artificial flanking regions, and inserted into the plasmid with the other chimeric cluster miRNA for artificial cluster generation (Fig. 1) according to the manufacturer's instructions.

Bottom Line: Here we show that the substitution of chimeric sequences with CHO-specific sequences for expression of miRNA clusters yields significantly higher expression levels of the mature miRNA in the case of miR-221/222 and miR-15b/16.Our data suggest that the Drosha/Dgcr8-mediated excision from primary transcripts is reduced for chimeric miRNA sequences compared to the endogenous sequence.See accompanying commentary by Baik and Lee DOI: 10.1002/biot.201300503.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Boku University Vienna, Austria; ACIB GmbH, Austrian Centre of Industrial Biotechnology, Graz, Austria.

Show MeSH