Fig 1.
Cloning of human erythropoietin.
(A) Illustration of different primer combinations used for amplification and cloning of EPO gene. The initiation (ATG) and termination (TGA) codons of translation are represented in bold characters. (B) Schematic representation of the T-DNA segments in EPO expression constructs with or without cal signal sequence (pK7cal/rhEPO or pK7rhEPO respectively). The transcription of EPO gene is controlled by P35S promoter and T35S terminator. EGFP-ER is transcriptionally controlled by Prol D promoter and T35S terminator. The cal signal peptide is fused to the N-terminus of EPO in the pK7cal/rhEPO construct.
Fig 2.
Agrobacterium rhizogenes mediated transformation and growth kinetics of the hairy root cultures.
(A) Hairy root culture with cal signal sequence pK7cal/rhEPOHR, hairy root culture without cal signal sequence pK7rhEPOHR and control hairy root cultures Nt/ATCCHR were shown. Scale bars are 1 cm. (B) Establishment of the initiated hairy roots (1–2 cm) in 50 mL of liquid WPM. Scale bars are 5 cm. (C) Graphical representation of growth kinetics for the hairy root cultures. All samples were measured in triplicates and each data represents the average ± SD.
Fig 3.
Regeneration of T0 tobacco plantlets.
(A) The 2 week old transgenic or control calli were cultured in regeneration medium. Scale bars are 5.8 cm. (B) The 4 week old transgenic or control regenerated T0 plantlets (pK7cal/rhEPORP or Nt/ATCCRP respectively) were grown in MS medium. Scale bars are 5.9 cm.
Fig 4.
GFP expression in hairy root cultures and in leaves of regenerated plant.
(A) Confocal images obtained from 15 d old hairy root cultures. (B) Confocal images obtained from 4 week old leaf tissues of regenerated T0 transgenic plantlets. Images are representative for three experiments and the scale bars are 50 μm.
Fig 5.
Genomic DNA and transcript analysis of hairy root cultures.
(A) Southern blot analysis was performed using the genomic DNA of transformed hairy root clones. (B) RT-PCR analysis of EPO mRNA in hairy root cultures pK7cal/rhEPOHR and pK7rhEPOHR. The presence of mRNA for tobacco β actin gene is shown as an internal control for transcript analysis.
Fig 6.
Western blot and ELISA analysis for effect of PVP treatment on the expression or yield of rhEPO.
(A) Western blot analysis of intracellular and extracellular total protein was performed for 20 d old hairy root cultures following the addition of PVP to the growth medium. For size comparison, recombinant human erythropoietin rhEPOCHO of molecular size 37 kDa from CHO cells (Sigma) was used. (B) ELISA analysis of extracellular rhEPOHR concentration in hairy root culture pK7cal/rhEPOHR is shown as ng ml-1 for three different intervals 10, 20 and 35 d. (C) ELISA analysis of intracellular rhEPOHR concentration for 20 d old hairy root culture pK7cal/rhEPOHR is shown as ng/μg of total soluble protein is shown as ng μg-1 of total soluble protein. (D) ELISA analysis of intracellular rhEPORP concentration in 4 w old leaf tissues of transgenic plants pK7cal/rhEPORP regenerated from hairy root cultures pK7cal/rhEPOHR is shown as ng μg-1 of total soluble protein. Each bar represents the average ± SD in panels B–D. Student’s t-test was performed to analyze significance, N = 3, *** and +++ p<0.001, ** and ++ p<0.01, and n.s. indicates no significance in panels B–D.
Fig 7.
HPLC profile of rhEPO in transformed and control hairy root cultures.
(A) Positive control rhEPOCHO standard recombinant human erythropoietin from CHO cells. (B) Hairy root culture pK7cal/rhEPOHR. (C) Negative control hairy root culture Nt/ATCCHR. The arrow indicates the presence of peaks.
Fig 8.
Bio-assay of rhEPO in a retinal pigmented epithelial (ARPE) cells.
(A) Impact of rhEPOHR on viability in ARPE cell lines was measured with MTT assay. (B) Impact of rhEPOHR on proliferation of ARPE cells was analysed with BrdU cell proliferation assay. The results are presented as percentage of cell viability in Panel A and percentage of cell proliferation in panel B. Student’s t-test was performed for Panels A and B to analyze significance, N = 3, *** p<0.001 and ** p<0.01 as compared to the corresponding data of untreated group (grey bars).