Silkworm eggs: An ideal model for studying the biological effects of low energy Ar+ ion interaction in animals

The object of the current work was to study low energy Ar + ion beam interactions with silkworm eggs and thus provide further understanding of the mechanisms involved in ion bombardment-induced direct gene transfer into silkworm eggs. In this paper, using low-energy Ar + ion beam bombardment combined with piggyBac transposon, we developed a novel method to induce gene transfer in silkworm. Using bombardment conditions optimized for egg-incubation (25 keV with ion fluences of 800 × 2.6 × 10 15 ions/cm 2 in dry state under vacuum), vector pBac{3 × P3-EGFPaf} and helper plasmid pHA3pig were successfully transferred into the silkworm eggs. Our results obtained from by PCR assay and genomic Southern blotting analysis of the G1 generations provide evidence that low-energy ion beam can generate some craters that play a role in acting as pathways of exogenous DNA molecules into silkworm eggs. Keywords Low-energy Ar + ion beam Silkworm eggs piggyBac transposon Transgenic silkworm PCR Inverse PCR Southern blotting analysis 1 Introduction The increasing travelling by air leads to an increase in the number of persons exposed to ionizing radiation called cosmic radiation, which include pilots, cabin crews as well as passengers [1] . Besides, predictions of the nature and magnitude of risks posed by exposure to radiation in space are subject to many uncertainties. Exposure to space radiation is an important barrier to exploration of the solar system by human beings because of the biological damage produced by cosmic radiation [2] . Hence, studying the biological effects of ion irradiation will facilitate the development of efficient countermeasures to radiation damage. The low-energy ion beam biotechnology uses low-energy ion beam to bombard biological organisms to induce biological effects. The effects have been applied successfully for mutation breeding and gene transfer in plants and microbes with high efficiencies. However, there were no reports of the successful application in transgenic animals. Moreover, the mechanisms of action are not well clarified. Bombyx mori provides an attractive alternative to other animals as a host organism since it is the domesticated silkworm, easy to rear, adaptable to continuous incubation and propagation, representing a major insect model for genetics and mutation research. It is the first lepidopteran for which draft genome sequences became available in 2004 [3] . Moreover, the silkworm eggs, deposited adherently and orderly, are favorable to the bombardment. The genetic transformation of silkworm was performed more than 20 years ago [4] , opening new possibilities for the insertion of exogenous genes into this organism. Since then, many different silkworm transformation procedures have been developed. By scanning electron microscopy (SEM), some craters, or ‘pimple-like’ structures, with a diameter of approximately 100 nm can be observed on the inner and outer surface of low ion beam-bombarded silkworm egg shell, obviously piercing the egg through the entire egg shell [5,6] , and providing the essential prerequisite for the low-energy ion beam induced gene transfer. All of the above have made it possible to carry out some investigations on low-energy ion beam induced DNA transfer in silkworm eggs. Here, we report the use of low-energy Ar + ion beam bombardment combined with piggyBac transposon technique to explore the effects of low energy Ar + ion interaction with silkworm eggs, which represents a novel silkworm transformation method for studying the potential mechanisms of ion bombardment-induced gene transfer in silkworm eggs. 2 Experiment 2.1 B. mori strain and eggs preparation for bombardment Non-diapause eggs of N4, a multivoltine B. mori variety, were used in this study. Silkworm larvea were reared at 25 °C and fed with mulberry leaves from spring to autumn and artificial diet during winter. The day before bombardment, male and female moths were allowed to mate through the night. In the morning, the females were placed on filmy paper and allowed to lay for 1 h. The eggs were disinfected with 75% ethanol solution for 3 min, then followed with absolute ethanol to dry. The piece of paper with eggs was put into a 60 mm Petri dish. Eggs were bombarded no later than 2 h after oviposition. 2.2 Parametric model of low-energy Ar + ion beam bombardment By the low-energy ion beam bioengineering facility (LEIBBF) ( Fig. 1 ), at 25 keV to fluences of 800 × 2.6 × 10 15 ions/cm 2 in dry state under vacuum (2 × 10 −2 to 8 × 10 −3 Pa) [5] , inert argon ions bombarded the silkworm eggs and stopped for ten seconds per 50 × 2.6 × 10 15 ions/cm 2 fluences. And then the eggs were immersed in 4 ml of a 1:1 (0.5 mg/ml total DNA concentration) mixture of vector pBac{3 × P3-EGFPaf} and helper plasmid pHA3pig in deionized water immediately [7,8] , and immersed more than 6 h at 25 °C. 2.3 EGFP expression detection Under an inverted Olympus IX70 fluorescence microscope, G0 eggs were screened for EGFP expression from the fifth day of incubation onwards. After hatching from bombarded eggs, first instar larvae were fed with artificial diet and reared in groups at 25 °C. G0 adults were mated together and G1 eggs were screened for EGFP expression. There were four types of controls analyzed in this transgenic procedure: (1) ion beam bombarded control (exposed but not immersed with plasmid DNA solution), (2) vacuum control (unexposed), (3) solution control (unexposed but immersed with plasmid DNA solution) and (4) blank control. 2.4 PCR detection and statistical analysis of integration efficiency Genomic DNA was extracted from G0 and G1 moths. DNA was incubated with proteinase K and then purified by standard SDS lysis-phenol extraction treatment. The total DNA was amplified using Taq polymerase under standard conditions with the primer pair 5′-TCATGGTGAGCAAGGGC-3′ and 5′-CTCACTTGTACAGCTCATCC-3′ designed from the 5′ and 3′ regions of the gfp gene. PCR was performed with a 5 min denaturing cycle at 95 °C followed by 35 cycles of 40 s at 95 °C, 30 s at 55 °C, 40 s at 72 °C, and a final extension at 72 °C for 10 min. PCR products were separated by 1% agarose gel electrophoresis and the transgenic integration efficiency was figured according to the number of single bands. 2.5 Inverse PCR (iPCR) and insertion site determination Genomic DNA was extracted as above from G1 moths. DNA was digested with Hae III [7] and circularized by ligation for 3 h at 16 °C using T4 DNA ligase. PCR was performed on the circularized fragments with primers designed from the left and right flanking of the piggyBac vector. For the left flank, the forward primer 5′-CTTGCACTTGCCACAGAGGACTATTAGAGG -3′ and reverse primer 5′-CAGTGACACTTACCGCATTGACAAGCACGC-3′ were used. For the right flank, the forward primer 5′-CCTCGATATACAGACCGATAAAACACATGC-3′ and reverse primer 5′-AGTCAGTCAGAAACAACTTTGGCACATATC-3′ were used [7] . PCR products were separated by electrophoresis in 1% agarose gel and plugs of single bands were purified (Spin Column DNA Gel Extraction Kit, Sangon) and sequenced after cloning in pMD19-T. Sequences were analyzed using Basic Local Alignment Search Tool (BLAST) searches of NCBI databases. 2.6 Southern blotting analysis and insertion analysis Genomic DNA was extracted as above from G1 moths. DNA was digested with Hae III and blotted onto nylon filters (Hybond N + , Pharmacia, America). The probe was generated by PCR (PCR DIG Probe Synthesis Kit, Mylab) using the primer pair 5′-TCATGGTGAGCAAGGGC-3′ and 5′-CTCACTTGTACAGCTCATCC-3′ and gfp gene as template. Hybridization was carried out at 65 °C in Hyb-50. Washing was done at room temperature in 2 × SSC and 0.1% SDS, followed by a 15 min wash at 50 °C in the same solution, 15 min at 50 °C in 1 × SSC and 0.1% SDS, and two 15 min washes at 50 °C in 0.1 × SSC and 0.1% SDS. The DIG-labeled hybrids was examined by color detection method (DIG Nucleic acid Detection KitI: Kit for color detection with NBT/BCIP, Mylab). 3 Results and discussion In this study, we have developed a novel method of generating transgenic silkworms and, for the first time, demonstrated that low-energy argon ion beam bombardment can induce piggyBac transposon transfer in living silkworm eggs with relatively low radiation damage. 3.1 Evidence for hereditary integration of g f p gene We bombarded 826 eggs, from which 751 larvae hatched (91%), which gave a hatching frequency 2.6 times higher than that for traditional microinjection [7] . As shown in Fig. 2 , 1–12 individuals were hatched from the bombarded and immersed eggs as detected by PCR analysis. As expected, a unique amplified band (750 bp) was observed, indicating that the gfp gene indeed existed in the examined individuals. However, in the control sample 13–16, none of the expected amplified products was detected ( Fig. 2 ). We detected 160 G0 moths, from which objective band amplified in 61 individuals in the PCR assay. Thus the percentage of transgenic integration efficiency was 38%, which obviously is better than that obtained from any other method in gene transfer [7] . Our data show that, as a method to deliver DNA, low-energy ion beam bombardment is highly efficient since thousands of silkworm eggs can be bombarded in less than 10 min, which proves to be much more rapid than other methods [9] . Inverse PCR experiments were performed on G1 progeny in order to verify that the insertion of the transgenes was due to transposition events. Genomic sequences were examined at the insertion sites. The sequence of the piggyBac inverted terminal repeats was recovered and bordered by TTAA sequence, the known target site of piggyBac . Four different integration events were identified ( Table 1 ). The insertions were flanked by sequences of piggyBac donor vector pHA3pig DNA, except that of the No. 4 insect in which surrounding genomic DNA was 100% identical to the sequence in B. mori chromosome 6 (Basic Local Alignment Search Tool, NCBI databases). The presence of vector DNA in the genome of transformed silkworms was determined by Southern blot analysis of DNA isolated from G1 positive individuals, using the total GFP-encoding sequence as a probe. Six of the insects were found to carry two insertions whereas two had three insertions ( Fig. 3 ). All eight DNA samples contained pBac{3 × P3-EGFPaf} hybridizing sequences, while DNA from controls did not have the sequences. 3.2 EGFP expression analysis Although no induction of the pBac{3 × P3-EGFPaf} vector mediated green fluorescent protein fluorescence was detected in this study, the results of three molecular biological methods for detecting exogenous gene integration were positive. There are many factors that influence the expression of foreign genes, such as integration site, introduction time (after bombardment) and DNA concentration. The effective expression is a puzzling question for researchers in plant or animal genetic engineering. 3.3 The possible mechanisms of plasmid DNA introduction into silkworm eggs We have pointed out in previous publication [5] that the hatching rates decreased with the increase in the fluence and energy level, and Ar + ion beam with energy, at a certain range of fluence, are able to generate etching effects on the eggshell surface. Ion beam application in life sciences is based on the hypothesis of a combination of energy absorption, mass deposition, and charge transfer of energetic ions in the organisms [10,11] . Our study provides supportive evidence for this hypothesis. (1) A silkworm eggshell has a porous lamellar structure. The eggshell thickness about 10 μm is naturally inhomogeneous. Therefore, it is entirely possible for low energy ion beam to pierce the eggshell by etching and ion channels effects [5,10] . But whether the ions really penetrated the eggshell requires further research. The eggshell firmness is maintained by a large number of hydrogen bonds, metallic bridged bonds and disulfide bonds that form between the chorion protein molecules. The implanted ion energy deposition may destroy the bonds and cause molecules and groups to be lost from the surface and even whole molecules to be broken up. These molecular fragments may escape from the eggshell and be pumped out from the vacuum. Consequently, the eggs in the vacuum chamber suffered severe shrinkage due to their contained water loss [12,13] . The energetic ion collisions may induce atomic displacement cascades, and the displaced atoms may recombinate with background elements. The result of these reactions indicate that the eggshell is pierced, forming a micro-hole. The plasmid DNA could transfer into eggs through the micro-holes [5,14–18] . (2) The charge transfer between the positive Ar + ions and the surface of an eggshell can cause decreased electrostatic repulsion force on foreign DNA. At the same time, the positive charge of the implanted ions accumulates on the inner wall and the bottom of a micro-hole, since the eggshell is a poor conductor of electricity. Thus, foreign DNA with the negative charge is relatively easy to transfer along the micro-holes into the silkworm eggs. (3) Some dissected and inactive chromosomes by a ion beam bombardment process stimulate the DNA rejoining and repair capacity, which increase the insertion and integration opportunities of foreign DNA [19] . 4 Conclusion In this study, our results show that it is possible to induce successful plasmid DNA transfer into silkworm eggs using low-energy ion beam bombardment with appropriately moderate argon ion fluence, accelerated with 25 keV (optimized for egg-incubation and etching effect), which has a high rate of transgenic integration for plasmid concentrations around 0.5 (mg/ml). Although no induction of the pBac{3 × P3-EGFPaf} vector mediated green fluorescent protein fluorescence was detected in this study, three molecular biological methods have demonstrated the exogenous gene integration convincingly. Integration site and introduction time (after bombardment) are the main factors that affect the expression of exogenous gene. Since the ion beam bombardment can be completed in less than 10 min this method is much easier and faster than other silkworm transformation methodologies. Moreover, our study provides new evidence for the hypothesis on the mechanisms of ion irradiation induced DNA transfer. With the establishment of ion beam bombardment as a novel method for rapid and effective gene transfer in silkworm with a minimal amount of experimental complexity, further investigations will be carried out to provide new insights in obtaining better expression of foreign genes and application of low energy ion irradiation to transgenic animals, which may enhance the investigations on how low energy Ar + ion interacts with complicated organisms. Acknowledgements We greatly acknowledge National Natural Sciences Foundation of China (No. 10975002 ) and the help of the Institute of Plasma Physics, the Chinese Academy of Sciences, for the use of its low-energy ion beam bioengineering facility (LEIBBF). 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