Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes

Lacy‐Hulbert, Adam; Thomas, Richard M.; Xp, Li; Ce, Lilley; Rs, Coffin; Roes, Jürgen

doi:10.1038/sj.gt.3301440

Cited by 44 publications

(39 citation statements)

References 20 publications

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“…Two antisense copies of either the 5'-or the 3'-arm of a particular mirtron were cloned region into two exons. 23 Proper splicing and expression of the modified EGFP (EGFPm) were confirmed by using an 81 nt long mouse intron and its 5' splice site mutated control in the coding can function in a context-independent manner, we tested the expression of human mirtrons from a heterologous coding environment. We modified the EGFP sequence to separate its coding for mir-1226sm.…”

Section: Resultsmentioning

confidence: 99%

“…28 For expressing mirtrons from a heterologous sequence environment, first a PvuII restriction site was introduced into the EGFP sequence by site directed mutagenesis. 23 Oligonucleotides corresponding to the sense and antisense sequence of the specific mirtrons were hybridized to form a double-stranded DNA, then inserted as an artificial intron into the PvuII site of EGFP by blunt end ligation. To generate splicing controls, oligonucleotides with mutated 5' splicing sites (GT > TG) were used.…”

Section: Methodsmentioning

confidence: 99%

“…To generate splicing controls, oligonucleotides with mutated 5' splicing sites (GT > TG) were used. For non-mirtronic controls, the third intron of mouse IgCε gene 23 and the human SREBF1 gene intron containing mir-33b were PCR amplified from genomic DNA samples and inserted into the PvuII site of EGFP.…”

Section: Methodsmentioning

confidence: 99%

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Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner

2012

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner

2012

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“…20 ); the underlying mechanism is unknown but might be due to increase in stability of the fusion protein formed after splicing. Another application includes studying RNAi by targeting the cloned small interfering RNAs (siRNA) against the red fluorescent protein 21 .…”

Section: Discussionmentioning

confidence: 99%

Cloning of intronic sequence within DsRed2 increased the number of cells expressing red fluorescent protein

Pisal¹,

Hrebíková²,

Chvátalová³

et al. 2017

BIOMED PAP

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“…This approach would create a gene structure reminiscent of typical mammalian genes to provide a nearnatural substrate for gene expression. 28 In our bioengineering strategy, 5 hFIX-expressing plasmids equipped with different combinations of 2 hBG introns inside the hFIX-cDNA and Kozak element ( Fig. 1) were used for expression analysis in HepG2 and CHO cells.…”

Section: Introductionmentioning

confidence: 99%

Bioengineering of differentiated hepatocytes withhuman factor IX-expressing plasmidsin vitro

2016

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For somatic gene therapy of hemophilia B, hepatocytes as the main cellular host for expression of hFIX are attractive targets. In gene therapy protocols, an efficient expression vector equipped with cis-regulatory elements such as introns is required. With this in mind, hFIX-expressing plasmids equipped with different combinations of 2 human b-globin (hBG) introns inside the hFIX-cDNA and Kozak element were used for bioengineering of HepG2 cells as a model for differentiated hepatocytes and CHO cells a cell line generally used to produce recombinant hFIX (rhFIX).In HepG2 cells, the highest hFIX secretion level occurred for the intron-less plasmid with 8.5 to 53.8-fold increases, while in CHO cells, the hBG intron-I containing plasmid induced highest hFIX secretion level with 2.3 to 14.3-fold increases as compared to other plasmids. The first hBG intron appears to be more effective than the second one in both cell lines. The expression level was further increased upon the inclusion of the Kozak element. The highest hFIX activity was obtained from the cells that carrying the intron-less plasmids with 470 mU/ml and 25 mU/ml for HepG2 and CHO cells respectively. Secretion of active hFIX by all constructs was documented except for hBG intron-II containing construct in both cell lines. HepG2 cells were able to secret higher hFIX levels by 0.6 to 112.2-fold increases with activity by 5.3 to 16.4-fold increases compared to CHO cells transfected with the same constructs. Presence of both hBG intron-I and II inside the hFIX-cDNA provides properly spliced hFIX transcripts in both cell lines.In conclusion, the advantages of hBG introns as attractive cis-regulatory elements to obtain higher expression level of hFIX particularly in CHO cells were demonstrated. Hepatocytes could be effectively bioengineered with the use of plasmid vectors and this strategy may provide a potential in-vitro source of functional hepatocytes for ex-vivo gene therapy of hemophilias and production of rhFIX in vitro.

show abstract

Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes

Cited by 44 publications

References 20 publications

Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner

Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner

Cloning of intronic sequence within DsRed2 increased the number of cells expressing red fluorescent protein

Bioengineering of differentiated hepatocytes withhuman factor IX-expressing plasmidsin vitro

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