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preface
In many organisms, specific gene functions have been successfully suppressed by gene silencing or editing, however, genetic manipulation to suppress target tissue function has not been achieved using cytotoxic genes. Here we established transgenic silkworms with posterior silk glands (PSGs) expressing the enzymatic domain of cytotoxin pierisin-1A (P1A) to validate that novel methods by targeting P1A expression can be used to develop biologically useful model organisms with tissue-specific dysfunction.
Expression and physiological effects of truncated P1A in silkworm PSG
The cabbage butterfly-derived P1A has 75% of the same amino acids as the apoptosis-induced humoral protein Pierisin-1, and the in vitro translated P1A protein exhibits ADP ribosylation activity, manifested by the transfer of ADP ribosylation of the substrate [32P]NAD to DNA; However, HPLC analysis of the reaction products showed that the DNA ADP ribosylation activity of P1A was 5%∼ (P < 0.001) compared to pierisin-1.
Transfection experiments were performed with a vehicle, and then immunoblotting was used to confirm that the N-terminal fraction of P1A contained the DNA ADP ribosyltransferase domain (P1A269) in an insect-cultured cell line, i.e., B. Transient expression in mori-derived BM-N and Spodoptera frugiperda-derived Sf21.
Ectopic gene expression of P1A269 and full-length P1A exposes a unique non-apoptotic effect on BM-N cells, characterized by an increase in the number of flat cells and inhibition of reporter protein synthesis.
To explore the functional consequences of intracellular expression of P1A269 in specific tissues, we generated the transgenic silkworm line w1-pndP1A269/P1A269, which carries a homogeneous transgene expressing P1A269 under the control of the fibrin heavy chain (FibH) promoter, which is particularly active in PSG. The role of P1A269 is expected to be limited to PSG because it is designed to lack both the signal sequence required for P1A secretion from PSG cells and the C-terminal receptor binding domain required for its invasion of target cells.
Reverse PCR and DNA database analysis revealed that the transgene was inserted into chromosome 16, and by using western blotting and SDS/PAGE, the expressed P1A269 protein could be detected in larval PSG on day 6 of the fifth instar, similar to control samples isolated from cyclic virus polyhedral coated P1A269 and P1A269-expressing Sf21 and BM-N cells.
In the larval stage, protein expression of P1A269 increases on day 4 of the fourth instar and on days 3 and 6 of the fifth instar, but decreases on day 7 of the fifth instar. Quantitative RT-PCR also showed that mRNA expression of P1A269 increased on days 3 and 6 of the fifth instar.
PSGs dissected from w1-pndP1A269/P1A269 larvae did not die due to apoptosis, but exhibited morphological abnormalities such as depression compared to PSG of untransformed w1-pnd+/+ larvae. In this study, the fifth instar larvae of w1-pndP1A269/P1A269 began spinning on day 6 of the fifth instar and continued to rotate vigorously after intestinal cleansing on day 7.
Previous studies have shown that FibH promoter activity in PSG increases with pulsations of ecdyhormone secreted by larvae on day 3 of the fifth instar and later in the fifth instar. On day 4 of the fourth instar, the larvae in this study are considered to be in the molting phase, when ecdyhormone pulses affect the expression of certain genes.
Therefore, the expression of P1A269 on day 4 of stage 4 larvae and days 3 and 6 of stage 5 larvae may be controlled by ecdystropin secretion, suggesting that ecdysin pulses repeatedly induce the expression of P1A269 during larval development, which is the reason for the abnormal appearance of PSG.
Genetic traits of w1-pndP1A269/P1A269 silkworms
The thin-layered cocoon shell produced by w1-pndP1A269/P1A269 larvae is 79% less weight than the cocoon shell produced by w1-pnd+/+ larvae (P < 0.001), and the cocoon shell of wild-type silkworms is composed of 70% fibrin [FibH and fibrin light chain (FibL)], 25% sericin and other materials.
Fibrin is a raw silk component produced exclusively from PSG, while sericin is a colloidal protein produced mainly by the intermediate silk glands that promotes cocoon cocoon cohesion by enveloping and sticking fibrin threads together. Therefore, the reduced weight of cocoon shells produced by w1-pndP1A269/P1A269 larvae is thought to be most likely due to reduced fibrin content.
SDS/PAGE in a gradient gel (5-20%), followed by western blotting with FibL-specific antibodies, shows that FibL and FibH proteins are present in the cocoon shells of w1-pnd+/+ larvae, but not in the cocoon shells of w1-pndP1A269/P1A269 larvae, where only nonfibrin, mainly serine, can be detected (serine 1-4).
Quantitative RT-PCR analysis showed that the mRNA levels of FibH and FibL in the PSG of fifth-instar larvae decreased strongly on days 5, 6, and 7 relative to w1-pndP1A269/P1A269 larvae. In fact, on day 7 of the fifth phase, the FibH and FibL mRNA levels of w1-pndP1A269/P1A269 larvae decreased to 3.1% and 0.9% of the W1-pnd+/+ larvae FibH and FibL mRNA levels, respectively (P < 0.001).
mRNA levels in the PSG of w1-pndP1A269/P1A269 larvae do not appear to decrease on the same day relative to the cytoplasmic A3 level of w1-pnd+/+ larvae. These results showed that intracellular expression of P1A269 inhibited the synthesis of FibH and FibL proteins in the PSG of transgenic silkworms.
The average weight of male and female w1-pndP1A269/P1A269 pupae on the third day after pupalation was 1.5 and 1.3 times higher than that of male and female w1-pnd+/+ pupae, respectively (P < 0.001).
This extra weight may be due to the retention of nutrient resources originally used for protein production, especially cellulose, which is needed for silkworm cocoon spinning. In fact, in classic experiments, surgical removal of the silk glands of the fourth and fifth star larvae causes the accumulation of excess body fluid amino acids, such as Gly, Thr, Ser, and Tyr, before pupation, while the larvae that remove the entire silk gland fail to pupate, possibly due to the presence of excess amino acids.
w1-pndP1A269/P1A269 pupals did not show obvious developmental defects after pupation; So far, these transgenic silkworms can mature to adulthood and produce healthy offspring that can reproduce successfully. These results suggest that nutrient resources reserved for cellulose synthesis do not appear to have a negative impact on transgenic silkworms.
Non-cocoon-shaped silk threads spun during the first to fourth molting are known to hold the larval's original legs to the surface, a behavior that is essential to support larval molting.
In this study, spun cellulose-free non-cocoon silk larvae did not peel. This result is consistent with evidence from Takasu et al. that the non-cocoon silk of wild-type silkworms that support larval molting contains little cellulose, but is composed mainly of sericin components, specifically sericin-2.
Application of the P1A269 allele in silkworm breeding
We next established the KWP1A269/P1A269 strain, which mates commercial silkworm strains (Kinshu x Showa) used in sericulture with w1-pndP1A269/P1A269 strains, screens individuals with genetic markers (EGFP-positive eyes), and then performs continuous sibling mating. This commercial silkworm strain produces heavier cocoons than W1-PND+/+.
The resulting KWP1A269/P1A269 larvae produce sericin cocoon shells in which FibL and FibH proteins are undetectable by SDS/PAGE and FibL-specific western blotting.
In addition, the cocoon shell of KWP1A269/P1A269 is 1.7 times heavier than that of w1-pndP1A269/P1A269 (P < 0.001), which results indicate that the w1-pndP1A269/P1A269 strain is simply mated with the preserved strain described in the silkworm genetic resources database (shigen.nig.ac.jp/silkwormbase/top.jsp) that is known to produce heavy cocoons. It can be used to produce cellulose-free heavy silkworm cocoons
Sericin cocoons can be used to prepare hydrogels
Commercial sericin prepared by degumming raw silk from common silkworm cocoons cannot form hydrogels because the high temperature and alkaline pH conditions during the degumming process cause protein decomposition, so that sericin can no longer be detected by SDS/PAGE. However, intact, soluble, cellulose-free serum proteins, whose concentrations can be detected by SDS/PAGE, can be easily prepared from w1-pndP1A269/P1A269 serum protein cocoons using 6M LiBr.
Intact, soluble mitomycin formation can be induced after 1 h of incubation with 10% ethanol, and it is not possible to obtain cellulose-free complete sericin solution from wild-type silkworm cocoons with the same chemical due to the high degree of contamination of cellulose in the extracted low-concentration sericin solution.
Intact soluble silk protein has similar moisturizing properties to collagen and can gellize in solutions up to 96% water, and intact sericin solutions form hydrogels on their own after long-term storage at 4°C for 2-3 weeks. After prolonged storage at 4 °C, slight alterations in the structure of sericin appear to be sufficient to promote gelation; Therefore, the addition of ethanol may have accelerated the process.
We previously showed that the activity of cytokines encapsulated in polyhedra, known as sericin microcrystals, can be maintained stably for a long time in vitro and in vivo environments and can be released slowly, continuously stimulating the growth and differentiation of multiple cell types.
Therefore, to determine whether sericin hydrogels can act as scaffolds to support cell growth and differentiation, we culture mouse embryonic stem (ES) cell line EB5 on sericin hydrogels covered with media containing typical recombinant human leukemia inhibitory factor (rhLIF) or adding polyhedral encapsulated LIFs.
EB5 cells that can remain undifferentiated in the presence of LIF can proliferate, form characteristic dome-shaped colonies, and express alkaline phosphatase (ALP). Differentiated EB5 cells cannot proliferate in medium containing blasticidin, which was used for all cultures in this study.
In control cultures grown on polyhedral sericin hydrogels or cultures incorporated into empty polyhedra, EB5 cells did not proliferate and formed ALP-stained positive colonies, and ALP activity extracted from cells grown on control hydrogels did not increase after culture.
In contrast, EB5 cells grown on polyhedral wrapped LIFs and EB5 cells grown on hydrogels covered with rhLIF-containing medium formed dome-shaped ALP-positive colonies with extracted ALP activity 14- and 18-fold higher than cells grown on control hydrogels with empty polyhedra added to rhLIF-deficient medium (P<0.005; Figure 3B); however, there was no significant difference between the two experimental groups (P>0.05). These findings suggest that sericin hydrogels can act as scaffolds to support EB5 cell growth as long as LIF is available.
Discussion of experimental results
This study yielded the transgenic silkworm w1-pndP1A269/P1A269, in which PSG expresses the DNA ADP ribosyltransferase structure of P1A, named P1A269. The successful expression of P1A in silkworm cells may be due to its low enzymatic activity relative to the highly cytotoxic Pierisin-1, which cannot be expressed in any kind of living cells.
Intracellular expression of P1A269 induced inhibition of reporter luciferase synthesis in BM-N cultured cells, and an increase in the number of cells with characteristic flat morphology was seen in BM-N cells expressing P1A269 transiently.
Thus, the response of BM-N cells to P1A269 appears to be related to cell cycle arrest and involves entering the quiescent phase, which is associated with impaired protein synthesis and activation of apoptotic pathways in response to DNA damage.
At the same time, P1A269 induced a decrease in FibH and FibL mRNA levels in PSG cells, PSG cells did not proliferate, but increased volume at the larval stage, indicating that P1A269 had the function of inhibiting the transcription of FibH and FibL genes.
Cytoplasmic actin A3 mRNA levels did not decrease due to the function of P1A269, indicating that P1A269 inhibited the transcription of some genes, including the fibrin gene that was initiated at the same time as the expression of the introduced P1A269. Therefore, it appears that the inhibition of some genes induced by P1A269 may be responsible for PSG cell growth and lead to observed morphological abnormalities.
The function of P1A269 appears to induce an increase in cytoplasmic actin A3 mRNA levels, however, since the function of P1A269 is estimated to inhibit a large increase in the transcription rate of rRNAs in PSG, which has been reported to occur simultaneously with fibrin synthesis, it is best to consider this result an unrealistic digital event from the normalization of data to the occurrence of P1A269 altered 18S rRNA levels.
So far, after treating mammalian cells with extracellular Pierisin-1, no cellular responses other than apoptosis have been observed because Pierisin-1 can enter target cells.
Therefore, only intracellular expression of P1A proteins is expected to induce characteristic cellular responses, including inhibition of cellular protein synthesis, suggesting that intracellular expression of P1A N-terminal domains can cause site-specific non-apoptotic effects that neither extend to neighboring tissues nor affect the developmental stage of the individual.
P1A can also be effective in any cell where it induces inhibition of protein synthesis. Thus, intracellular expression of P1A, which only encodes the active region of N-terminal enzymes, can be used to generate model organisms with tissue-specific dysfunction, suitable for a variety of genetic manipulations, including induction of quiescence in cancer cells in mammals, and the development of animal models of impaired islet function or impaired immune system function for diabetes research.
However, in order to apply some of the intracellular expression of P1A in other organisms in the future, we must first understand how P1A fragments induce PSG dysfunction by inhibiting protein synthesis rather than inducing apoptosis. One possibility is that a certain level of DNA ADP ribosylation in cells requires inhibition of protein synthesis, but above this level induces apoptosis.
The observation that P1A269 is expressed more strongly in Sf21 cells than in BM-N cells, where it stimulates its apoptosis cell death, supports this hypothesis. Therefore, future studies need to investigate whether the inhibition of protein synthesis that occurs in PSG depends on the strength of the expressed DNA ADP ribosylation activity, and how P1A activity is regulated by regulating promoters that control P1A269 expression or amino acid mutations that introduce P1A269.
The study also found that w1-pndP1A269/P1A269 larvae spun cocoons that contain almost no cellulose and consist almost entirely of sericin. A previous study showed that silkworm PSGs expressing the Bcl-2-associated X protein become ablated, although the study did not provide information about the development of manipulated individuals after cocoons and pupation.
In addition, a separate report describing T7 RNA polymerase-modified PSGs in transgenic silkworms showed reduced but detectable expression of FibL and FibH, while another study showed that thin-layer silkworm cocoons produced by silkworms with transcription activator-like effector nuclease-mediated disruption of the FibH gene did not contain FibH but had normal FibL levels.
Nd-silkworm strains have a natural genetic mutation that affects silk glands, producing small amounts of silkworm cocoons, which have a very high amount of sericin and varying proportions of cellulose. Our study reported the use of a cytotoxin that leads to the inhibition of PSG function to produce FibH and FibL, as well as the production of silkworm cocoons composed only of sericin.
epilogue
Through the study we can find that: in the future, further breeding can be carried out to produce transgenic silkworms with intermediate silk glands that produce sericin containing polyhedral encapsulated cytokines, from which pathogen-free artificial extracellular matrices can be created for in vitro and in vivo applications.
These silkworms can produce functional proteins in their silk glands that can serve as an effective, inexpensive alternative to collagen or other drug candidates for delivering biomaterials. Our results show that by targeting the expression of P1A, bioengineering has successfully produced silkworms with biologically useful traits with important application potential.
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