Insulin-Like Growth Factor 1 (IGF-1) stimulates the body’s systemic growth and has growth-promoting effects on almost all cells in the body, especially the liver, kidneys, skeletal muscle, bones, nerves, skin, and lung cells. Also, this factor, with its ability to bind to insulin receptors, has insulin-like effects and is involved in regulating DNA replication ( 2 ). IGF-1 is one of the most potent natural activators of the Protein Kinase B (PKB) signaling pathway, which is a stimulator of cell growth and proliferation and an inhibitor of programmed cell death ( 3 , 4 ). Because of the important functions of IGF-1 in the body, people who suffer from IGF-1 deficiency, often experience harmful side effects ( 5 ), which in its most severe form leads to a Laron-like dwarf phenotype ( 6 ). The efficacy and safety of IGF-1 therapy in these patients have been evaluated for more than two decades. There have also been reports in the past few years of the benefits of treating short stature in children with recombinant IGF-1 ( 7 ). For example, in Polish patients, treatment with recombinant IGF-1 had a clear effective and safe effect on patients’ height, especially in the first year of treatment ( 8 ).
Currently, most recombinant IGF-1 is produced using E. coli ( 9 ) or yeast ( 10 ), which are expensive. In addition, the formation of inclusion bodies in E. coli or the variable biological activity of IGF-1 produced in yeast is another disadvantage of current production systems. Plant systems have many advantages, including the low cost of large-scale plant production, the ability to easily store or transport, and the presence of natural organs for protein storage, suitable for the production of large-scale recombinant proteins on an industrial scale ( 11 ). The cost of plant-based recombinant proteins can be 10 to 50 times lower than the production of similar proteins in bacterial or yeast systems ( 12 ). However, a major drawback to the expression of human blood proteins through the nuclear genome is their low expression level, which is generally less than 1% of total soluble protein ( 13 ). For example, the expression level of IGF-1 in transgenic rice and tobacco, after optimizing codons for expression in plants and using optimal regulatory sequences, has been reported in the range of 0.002 - 0.011% of total soluble protein ( 14 ). Although progress has been made in recent years in increasing the expression of foreign genes ( 11 , 15 ), there are not many examples of high expression levels of human blood protein using nuclear genome transformation. In addition, the expression of foreign genes in the nuclear genome faces other challenges, such as the possibility of transgene silencing, the transgene position effect, and the existence of epigenetic effects ( 16 ). Over the past few years, with the transfer and expression of foreign genes in the chloroplast organelle, in addition to solving these problems, the expression of foreign protein has been reported to be more than 46% of leaf total soluble protein ( 17 ). Another important benefit of expressing foreign genes through the chloroplast genome is transgene containment in transgenic plants due to maternal inheritance of the chloroplast genome ( 18 , 19 ). In addition, therapeutic proteins expressed in transgenic chloroplasts have been demonstrated to be fully functional with proper post-translational modifications ( 20 ). Based on these advantages, several human blood proteins, including somatotropin ( 21 ), interferon gamma ( 22 ), interferon alpha ( 23 ), and insulin ( 24 ), have been expressed in Chloroplasts and their proper function has also been confirmed. In 2009, Henry Daniell and coworkers, examined the expression of native and synthetic versions of human IGF-1 in bacterial hosts (E. coli) and tobacco chloroplasts and showed that unlike the synthetic version with modified codon usage, the native version is not able to express in E. coli ( 25 ). The expression of recombinant protein was estimated to be about 11.3% of total protein, which could be increased up to 32% by optimizing plant growth conditions. Interestingly, unlike E. coli, the native and synthetic versions did not show much difference in chloroplast expression, indicating a high insensitivity of the tobacco chloroplast expression system to codon usage.
In this study, we examined the expression of native human IGF-1 in tobacco chloroplasts. Compared to the study done by Daniell et al. (2009) ( 25 ), in this study, the position of foreign gene insertion in the tobacco chloroplast genome, as well as factors controling IGF-1 gene expression were different, which will be discussed in the following.
3. Materials and Methods
3.1. Plastid-Specific Transformation Vector
The pRB94-IGF1 chloroplast vector ( 1 ) with a size of 7581 bp, which is a derivative of the pRB94 vector (Kindly provided by Prof. Ralph Bock, Max-Planck Institute for Molecular Plant Physiology, Golm, Germany) and carries mature IGF-1 cDNA with a size of 321 bp (Fig. 1A). In this vector, the aadA selectable marker gene is driven by the Prrn promoter (The tobacco plastid rRNA operon promoter) and the TpsbA terminator. The IGF-1 gene is also under the control of the Prrn promoter fused with the 5 ‘UTR of gene 10 from E. coli phage T7 and the downstream box (T7g10-DB), which have significant effects on RNA stability and translation efficiency ( 26 ), and the TrbcL terminator from tobacco. In addition, the Shine Dalgarno (SD) sequence, which is necessary to start translation in prokaryotic systems, was added just before the start codon. Transgene cassetes are cloned in an intergenic region between the trnG and trnfM genes of the tobacco chloroplast genome. The left flank (LF) region contains the trnG and psbZ sequences, and the right flank (RF) region contains the trnfM, rps14, and 3’ part of the psaB gene (Fig. 1). The expected physical map for the chloroplast genome (transplastome) after receiving the forein genes is shown in Figure 1B.
3.2. Transfer of Recombinant Vector to Tobacco Chloroplasts
BioRad PDS-1000/HeTM System gene gun was used to transfer recombinant plasmid to young leaf cells of tobacco plants (Nicotiana tabacum Petit Havana). In summary, young leaves were first cut from 3-week-old sterile tobacco seedlings and were placed on RMOP medium from their adaxial side (Fig. 2A). The abaxial side of the leaves were then bombarded with 0.6 μm gold particles coated with the pRB94-IGF1 vector with 1100 psi pressure. The leaves were then cut into 0.5 cm2 pieces and transferred to RMOP regeneration medium ( 27 ) containing 500 mg.L-1 spectinomycin. The cultures were placed in a growth chamber with a light cycle of 16 h light (25 °C) and 8 h dark (22 °C). After regeneration of potential transgenic plants and their initial confirmation using PCR, to obtain homoplasmic level, four rounds of selection and regeneration of leaf fragments from regenerated plants were performed. Last generation plants were used for further studies.
3.3. Examination of IGF-1 Transcripts by RT-PCR
RT-PCR was used to evaluate the IGF-1 expression in transgenic plants at the transcriptional level. In summary, the total RNA was extracted from leaf tissues using the RNXTM-Plus extraction kit (SINACLON, Iran) according to the manufacturer’s instructions. The samples were then treated with RNase-free DNase I to eliminate possible genomic DNA contaminations.
To prepare cDNA, after testing genomic DNA elimination by PCR on RNA samples, 14 μL of reaction solution containing 5 μg of the extracted RNA, 10 pM poly T oligonocleotide and 10 mM dNTP mixture, was placed at 65 °C for five min, followed by transferring on ice immediately. After one minute, 4 μL of 5× reverse-transcription buffer, 1 μL of 0.1 M DTT, 1 μL of 50 mM MgCl2 and 0.5 μL of Super Script III reverse transcriptase (5 U.μL-1) were added to the reaction solution. Synthesis of the first strand of cDNA was carried out for 1 hour at 50 °C. A pair of IGF-1 gene specific primers (5’-AAA CAT ATG GGA CCG GAG ACG CTC TG-3’ and 5’-AAA TCT AGA CTA CAT CCT GTA GTT CTT GTT TCC T-3’) were used to amplify transgene cDNA copies of 336 bp in size. PCR program included a 4-minute stage at 95 °C; 30 cycles, each consisting of 45 s at 95 °C, 45 s at 55 °C and 90 s at 72 °C; and a final extension at 72 °C for 10 min.
3.4. Investigation of the Physical Presence of Foreign Genes in the Chloroplast Genome Using Southern Blot
In addition to PCR, Southern blot was used to evaluate the physical presence of foreign genes in transgenic plants. To ensure the transfer of IGF-1 to the chloroplast genome, the plastid genome of transgenic plants in the region outside the Left flank and Right flank sequences was cut using the BglII restriction enzyme, in which case a 5528 bp fragment of the genomes of transgenic chloroplasts will be isolated (Fig. 1B). In summary, after extraction of whole DNA by cetyltrimethylammonium bromide (CTAB) method ( 28 ), about 10 μg of it was cleaved by BglII, separated by gel electrophoresis in 1% agarose gel, and transferred onto Hybond nylon membranes (Amersham, Buckinghamshire, UK) by capillary blotting using standard protocols.
The Biotin DecaLabel DNA Labeling Kit (Thermo Fisher ScientificTM) was used to hybridize and identify DNA fragments containing foreign genes based on the manufacturer’s description. The complete IGF-1 sequence, which was isolated from the vector by enzymatic digestion, was used as a probe (Fig. 1B).
3.5. Progeny Test to Evaluate the Successful Transfer of the Foreign Genes into the Chloroplast Genome
To investigate the inheritance of foreign genes, a two-way cross between wild-type and transgenic plants was used. Seeds from reciprocal crossing and selfing of transgenic and wild-type plants were grown on medium containing 500 mg.L-1 spectinomycin. In this medium, only seeds containing the aadA selectable marker gene will be able to germinate normally and produce green seedlings, while non-transgenic seeds form albino seedlings that are unable to continue growing in this medium.
3.6. Evaluation of IGF-1 Protein Expression by SDS-PAGE
Total soluble protein (TSP) was extracted from the leaf tissue of transgenic plants by the method described by Cahoon and coworkers ( 29 ) Approximately 50 μg of TSP was loaded in each well from SDS-PAGE gel. Proteins were stained on the gel using Coomassie Blue solution (0.1% Coomassie blue, 50% methanol and 10% acetic acid). Finally, the gel was decolorized with a solution containing 20% methanol and 10% acetic acid.
4.1. IGF-1 Gene Transfer by Biolistic Gun Method and Selection of Potential Transgenic Plants
The bombarded leaves (Fig. 2A) were cut in pieces and placed on medium containing 500 mg.L-1 spectinomycin. After about 4 weeks of selection, most of the leaf pieces bleached out and there were only a few green segments left, some of which regenerated into small seedlings after about 3 weeks (Fig. 2B). Of the 4 seedlings regenerated from 5 shoots using the hepta adapter, only one showed the desired band in PCR (Fig. 2C), and the rest were probably escapes or spontaneous mutations.
4.2. Creation of Homoplasmic Transplastomic Plants
Putative transgenic line was selected four generations on the regeneration medium containing 500 mg.L-1 spectinomycin to reach the homoplasmic transplastomic stage. In addition to PCR, to demonstrate successful transmission and expression of the aadA selectable marker gene in transgenic plants and to rule out the possibility of spectinomycin resistance due to spontaneous mutation, a test for resistance to another aminoglycoside antibiotic, including streptomycin, was performed at a concentration of 500 mg.L-1, (data not shown). Regenerated plants from the fourth selection round of transgenic line were transferred to the greenhouse for growth and seed production. These plants were phenotypically similar to wild type (Fig. 3A), and produced normal seeds.
4.3. Confirmation of IGF-1 Gene Expression at the Transcriptional Level
RT-PCR test was used to confirm the expression of IGF-1 gene at the transcriptional level in transgenic plants. The results of this experiment showed that unlike the wild type, the selected transgenic line contains transcripts of IGF-1 gene and as a result, the expression of this gene in transgenic plants is successful at the transcriptional level (Fig. 3B)
4.4. Investigation of Transgene Inheritance and Confirmation of Homoplasmy Through Progeny Test
To prove the transfer of the foreign gene into the chloroplast genome, its inheritance in the transgenic line was evaluated through reciprocal crosses. The results of this experiment showed that when wild-type plants are fertilized by the pollens of transgenic plant, the selectable marker gene is not transferred to the resulting progenies, and these seeds, like wild type, are sensitive to the spectinomycin antibiotic (Fig. 3C). Conversely, when the transgenic line was used as the maternal parent, all seeds became resistant to the spectinomycin, like the seeds derived from selfing of the transgenic line (Fig. 3C). These results indicate that the inheritance of the aadA selectable marker gene is cytoplasmic and the transgene has been successfully transferred to the plastid genome. It should be noted that the uniformity of transgenic plant seeds for spectinomycin resistance, as a sensitive method ( 27 ), indicates the degree of homoplasmy in transgenic plants.
4.5. Confirmation of IGF-1 Gene Insertion in to Plastid Genome by Southern Blot Method
To confirm the physical presence of IGF-1 gene in the chloroplast genome of transgenic plants and seeds resulting from their self-pollination, Southern blot analysis was used. According to the tobacco chloroplast genome map (NCBI accession number: NC_001879.2), after successful insertion of foreign genes at the designated site (Fig. 1B), by cutting the transgenic plastome with the BglII restriction enzyme, which cuts the transplastome in areas outside the flanking regions, it is expected to obtain a 5528 bp fragment containing foreign genes. Unlike the wild-type plant, the 4th-regeneration-round transgenic plant and its progenies showed the desired band in this experiment (Fig. 3D), confirming the successful insertion of the foreign genes into the chloroplast genome of the transgenic plant, and its transfer to the next generation offspring.
4.6. Evaluation of Transgenic Plant Proteins by SDS-PAGE Method
SDS-PAGE gel analysis was performed to evaluate the expression status of transgenic plant proteins and the possibility of identifying IGF-1 protein. The total soluble protein status of the transgenic plant was almost similar to that of the wild-type plant (Fig. 3E). However, in the region of about 12 kDa, where the size of IGF-1 protein is also in this range (12.8 kD), the accumulation of proteins was much higher than in the wild type, which is probably mainly related to the expression and accumulation of IGF-1 protein.
In this study, the transfer and expression of human Insulin-Like Growth Factor 1 (IGF-1) cDNA in tobacco chloroplasts was investigated. From 5 shots using the Hepta adapter, one true transformant was selected by PCR method, which is relatively acceptable for gene transfer to chloroplasts ( 18 , 30 ). Due to the precise transfer of foreign genes to a predetermined region in the chloroplast genome using the homologous recombination, there is no position effects in chloroplast transformation ( 18 ). Therefore, a single plastid transgenic line will be sufficient for further experiments. Four rounds of selection on regeneration medium containing high concentrations of spectinomycin (500 mg.L-1) eliminated all wild-type plastomes and replaced them with transplastomes, so that all seeds obtained from self-pollination of the transgenic plant were antibiotic resistant, which is a very important sign to achieve the homoplasmic stage ( 30 , 31 ). If the homoplasmic stage is not established, the wild-type plastid genome will multiply rapidly in the absence of selective agent, leading to removal of transgenic plastid genome copies and loss of antibiotic resistance very rapidly ( 30 ).
Integration of IGF-1 gene into the chloroplast genome was confirmed by two methods, including progeny test and Southern blot analysis (Fig. 3). Given that the chloroplast organelle is derived from prokaryotic ancestors ( 32 ), it is unlikely that the selectable marker gene will be expressed if transferred to the nucleus genome, although in rare cases it occurs by modifying the promoter or using adjacent nuclear gene promoters ( 33 ) Cytoplasmic maternal inheritance of the selectable marker gene in reciprocal crosses between the transgenic plant and the wild type, demonstrated the transfer of the selectable marker gene into the chloroplast organelle (Fig. 3C). These results were also confirmed using the Southern blot method (Fig. 3D).
RT-PCR analysis confirmed IGF-1 gene expression at the transcription level. Tobacco plastid rRNA operon promoter (Prrn) was used to control transcription of IGF-1 gene. This promoter has strong transcriptional activity with recognition elements for both Nuclear- (NEP) and Plastid- encoded (PEP) RNA polymerases ( 34 ). For this reason, the promoter has also been used to transcribe selectable marker gene to increase the chances for early transgenic cells to survive in the early stages of gene transfer to organelle, which a small number of transgenic plastid genome copies exist. In a study by Daniell et al. (2009), the PsbA promoter, which can only be recognized by the PEP RNA polymerase that is usually active in the presence of light, was used ( 25 ). Also, in this study, to increase IGF-1 protein expression, we used a modified version of the Prrn promoter, in which the 5’UTR of gene 10 from E. coli T7 phage and the downstream box (T7g10 DB) is fused to the promoter. This sequence has a significant effect on RNA stability and increased translation efficiency, and can lead to greater expression of foreign proteins ( 26 ). In the study of total soluble protein of transgenic plant by SDS-PAGE method, the protein accumulation was high in the range of 12 kDa, which is probably partly due to the high expression of IGF-1 protein with a size of 12.8 kDa. However, due to the accumulation of some natural wild-type proteins in this area (Fig. 3E), the detection of foreign proteins in this area is difficult and these results need to be confirmed by more accurate experiments such as Western blotting.
In this study, a natural version of human IGF-1 was used without modification of codon usage and its adaptation to tobacco chloroplasts. Daniell et al. (2009) also showed that the difference in IGF-1 expression between the synthetic codon-usage-optimized version and the natural version was negligible in chloroplast ( 25 ).
Cytoplasmic maternal inheritance of the chloroplast genome in genetically modified plants, as an efficient method for transgene containment and preventing its spread in nature, is valuable for the safe production of the transgenic crop in the field ( 35 ). In a few studies, the probability of foreign gene transmission through pollen grains has been reported to be around 2.86 × 10-6, which is a very low and insignificant probability ( 19 , 36 ). Because of such unique benefits, transgenic chloroplasts can have many applications as important biological reactors for the production of human pharmaceutical proteins ( 37 ). It should be noted that, appropriate biological activity of proteins produced by plant systems, including IGF-1, in human target cells has been reported in previous studies ( 13 , 15 , 25 , 37 ).
The Biotechnology Development Council and Azarbaijan Shahid Madani University are acknowledged for the financial support of this research. The author thanks Dr. Maghsoud Pazhouhandeh, Mona Bordbar, Neda Hassanpour and Maryam Bagheri for their assistance.
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