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Function and regulation domains of a newly isolated putative β-actin promoter from pacific white shrimp.

Shi Y, Soderlund M, Xiang J, Lu Y - PLoS ONE (2015)

Bottom Line: The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif.Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1.Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Current development of transgenic shrimp research has been hampered due to the lack of the suitable promoters and efficient transfection methods for crustaceans. A 1642 bp sequence, containing 5'-upstream sequence, exon 1, intron 1 and partial exon 2, which is responsible for transcriptional initiation of the newly reported shrimp β-actin (actinT1), has been isolated from the Pacific white shrimp (Litopenaeus vannamei) and named as SbaP. To determine its function and potential application in marine biotechnology, the sequence and functional domains were examined by constitutive expression of the luciferase reporter gene. We have identified 5' regions that play a central role in the expression of the β-actin gene. The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif. Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1. Transient transfection assay with a construct containing proximal promoter and enhancer (SbaPΔ-222/+1Δ-1325/-924) regions of the shrimp β-actin coupled with luciferase and EGFP (enhanced green fluorescent protein) showed that the promoter was not only functional in sf21 cells, but promoter activity was more than 8-fold higher than a viral-origin promoter (ie1, white spot syndrome virus immediate early gene promoter). Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

No MeSH data available.


Related in: MedlinePlus

Examination of activity of 5’-upsream sequences and 1st intron of the shrimp β-actin gene based on a reporter assay.A: schematic diagram of promoter region of luciferase reporter gene constructs. Showing various 5’-flanking sequence sequences of shrimp β-actin gene fused with luciferase gene. B: the relative levels of reporter gene expression in sf21 cells are shown. The constructs were transiently co-transfected into cells along with pRL-tk control vector. The activity of firely and Renilla luciferase in the cell lysate were measured using Dual-luciferase reporter assay (Promega) at 48h post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The Bars indicated mean ±S.D. of luciferase activity (n = 3).
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pone.0122262.g002: Examination of activity of 5’-upsream sequences and 1st intron of the shrimp β-actin gene based on a reporter assay.A: schematic diagram of promoter region of luciferase reporter gene constructs. Showing various 5’-flanking sequence sequences of shrimp β-actin gene fused with luciferase gene. B: the relative levels of reporter gene expression in sf21 cells are shown. The constructs were transiently co-transfected into cells along with pRL-tk control vector. The activity of firely and Renilla luciferase in the cell lysate were measured using Dual-luciferase reporter assay (Promega) at 48h post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The Bars indicated mean ±S.D. of luciferase activity (n = 3).

Mentions: To explore the promoter activity of newly isolated shrimp β-actin sequence, several constructs containing various regions of the 5’-flanking sequence were generated and fused to a luciferase gene. An initial simple construct contained all 1, 642 bp of 5’-flanking sequences (construct named pGL-SbaP, Fig 2A), which was compared with the luciferase reporter constructs composed of proximal promoter (pGL-SbaP-1642/-1115) and 1st intron (pGL-SbaP-1151/+1), respectively. Since cell lines derived from shrimp were not available, most of the current promoter studies were carried out under xenogenic conditions [19, 20]. The result indicated that the proximal promoter was able to drive luciferase gene expression successfully (Fig 2B). However, it showed a severe reduction compared to that of 5’-flanking sequence (pGL-SbaP), indicating there are regulatory elements within the 1st intron.


Function and regulation domains of a newly isolated putative β-actin promoter from pacific white shrimp.

Shi Y, Soderlund M, Xiang J, Lu Y - PLoS ONE (2015)

Examination of activity of 5’-upsream sequences and 1st intron of the shrimp β-actin gene based on a reporter assay.A: schematic diagram of promoter region of luciferase reporter gene constructs. Showing various 5’-flanking sequence sequences of shrimp β-actin gene fused with luciferase gene. B: the relative levels of reporter gene expression in sf21 cells are shown. The constructs were transiently co-transfected into cells along with pRL-tk control vector. The activity of firely and Renilla luciferase in the cell lysate were measured using Dual-luciferase reporter assay (Promega) at 48h post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The Bars indicated mean ±S.D. of luciferase activity (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4383542&req=5

pone.0122262.g002: Examination of activity of 5’-upsream sequences and 1st intron of the shrimp β-actin gene based on a reporter assay.A: schematic diagram of promoter region of luciferase reporter gene constructs. Showing various 5’-flanking sequence sequences of shrimp β-actin gene fused with luciferase gene. B: the relative levels of reporter gene expression in sf21 cells are shown. The constructs were transiently co-transfected into cells along with pRL-tk control vector. The activity of firely and Renilla luciferase in the cell lysate were measured using Dual-luciferase reporter assay (Promega) at 48h post transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The Bars indicated mean ±S.D. of luciferase activity (n = 3).
Mentions: To explore the promoter activity of newly isolated shrimp β-actin sequence, several constructs containing various regions of the 5’-flanking sequence were generated and fused to a luciferase gene. An initial simple construct contained all 1, 642 bp of 5’-flanking sequences (construct named pGL-SbaP, Fig 2A), which was compared with the luciferase reporter constructs composed of proximal promoter (pGL-SbaP-1642/-1115) and 1st intron (pGL-SbaP-1151/+1), respectively. Since cell lines derived from shrimp were not available, most of the current promoter studies were carried out under xenogenic conditions [19, 20]. The result indicated that the proximal promoter was able to drive luciferase gene expression successfully (Fig 2B). However, it showed a severe reduction compared to that of 5’-flanking sequence (pGL-SbaP), indicating there are regulatory elements within the 1st intron.

Bottom Line: The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif.Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1.Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Shandong, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Current development of transgenic shrimp research has been hampered due to the lack of the suitable promoters and efficient transfection methods for crustaceans. A 1642 bp sequence, containing 5'-upstream sequence, exon 1, intron 1 and partial exon 2, which is responsible for transcriptional initiation of the newly reported shrimp β-actin (actinT1), has been isolated from the Pacific white shrimp (Litopenaeus vannamei) and named as SbaP. To determine its function and potential application in marine biotechnology, the sequence and functional domains were examined by constitutive expression of the luciferase reporter gene. We have identified 5' regions that play a central role in the expression of the β-actin gene. The proximal promoter (-1642/-1325) contains two highly conserved transcriptional sites, CCAAT box and CArG motif. Two negative (-1140/-924, -222/-21) and one positive (-810/-425) regulatory elements have been identified in intron1. Transient transfection assay with a construct containing proximal promoter and enhancer (SbaPΔ-222/+1Δ-1325/-924) regions of the shrimp β-actin coupled with luciferase and EGFP (enhanced green fluorescent protein) showed that the promoter was not only functional in sf21 cells, but promoter activity was more than 8-fold higher than a viral-origin promoter (ie1, white spot syndrome virus immediate early gene promoter). Furthermore, SbaPΔ-222/+1Δ-1325/-924 drove a successful expression of luciferase injection assay in vivo injection and also showed higher promoter activity than the ie1 promoter, suggesting that the expression vectors constructed with SbaPΔ-222/+1Δ-1325/-924 have important potential in gene transfer studies for shrimp and other crustacean species.

No MeSH data available.


Related in: MedlinePlus