动物造模,穹窿蛋白 z-bio 2021-02-21 661

　　The evolutionarily highly conserved major vault protein (MVP) is the main component of the largest naturally occurring ribonucleoprotein. Studies have shown that MVP is involved in the regulation of a variety of cellular processes, including nuclear and cytoplasmic transport, signal transduction, cell differentiation, cell survival and immune response. Since most of the research is performed on in vitro systems, the physiological role of MVP is still largely unknown. Although the absolute function of the cell vault has not yet been determined, there are reports that these ribonucleoproteins may play a role in zebrafish organ regeneration. Expression analysis showed that the heart, caudal fin and spinal cord of zebrafish had MVP expression after injury. Using a morpholino-based knockout method, the researchers observed a reduction in axon regeneration after spinal cord transection, and proposed that MVP supports motor recovery and axon regeneration in adult zebrafish. In this study, we established a mvp gene knockout zebrafish strain to describe the function of mvp in the process of organ regeneration. mvp-/-zebrafish are feasible without obvious physical abnormalities. Data shows that lack of MVP will not cause significant changes in the regeneration capacity of zebrafish's heart, spinal cord and fins. Further experiments showed that in injured mvp knockout zebrafish, cell death increased significantly, while the transcriptome was basically unchanged. These data indicate that MVP plays an anti-apoptotic effect in the early stage of zebrafish injury response, but plays a negligible role in the regeneration of zebrafish. Mvp expression during regeneration: In order to better define the spatiotemporal expression pattern of mvp during regeneration, the mvp:EGFP reporter strain was constructed with the BAC sequence containing zebrafish mvp, and the EGFP in the damaged heart, spinal cord and fins was evaluated. signal. After resection of the apex, mvp-driven EGFP was induced in the entire dense layer of ventricular muscle within 24 hours. After 3 to 7 days, the transactivation of EGFP was restricted to the original layer of the myocardium, and then gradually closed. 24 hours after the adult zebrafish spinal cord transection operation, EGFP signal can be observed in the ependymal cells on the inner wall of the central canal. With the beginning of the regeneration process, the expression of EGFP briefly extended to neurons near the injury site, and mostly disappeared on the 7th day (dpi) after injury. Approximately 24 hours after caudal fin amputation, a bright EGFP signal appeared on the wound epidermis. During the process of blastocyst formation, mvp-driven EGFP is expressed in the blastoderm mesenchyme. During the regenerative growth phase, EGFP signal is suppressed in the newly formed non-mineralized needles on the distal edge of the fin. These data indicate that mvp is expressed in response to injury signals in the acute reaction stage, so MVP may regulate inflammation and cell survival in the early stages of multiple organ regeneration.

　　mvp gene knockout zebrafish: In order to clarify the function of mvp in regeneration, CRISPR-Cas9 was used to destroy the zebrafish mvp gene. Multiple sgRNAs targeting mvp sites were designed, transcribed and injected into 1-cell zebrafish embryos. The F1 generation was crossed with wild-type AB fish and screened for the deletion of the entire mvp coding region. The 22,490-bp allele deleted at the mvp locus was selected for subsequent analysis. In order to confirm the loss of mvp in the knockout line, the expression of mvp was detected by qPCR and a full set of in situ hybridization, which verified the absence of mvp mRNA in mvp-/- embryos. A number of mvp +/- incrosses embryos were collected and raised to the adult stage to study the role of mvp in zebrafish development, survival and fecundity. No morphological difference was observed between mvp-/- and wild-type animals. Similar to mvp gene knockout mice, no significant effects on overall survival and reproduction were observed in mvp-/- fish, so mvp mutant fish provide an opportunity to study its regeneration function.

　　The effect of mvp loss on organ regeneration: The most commonly used ventricular resection fish study analyzed the effect of mvp loss on heart regeneration. After injury, the zebrafish's heart responds to damage to the entire organ, and the epicardium is activated and plays an important structure and signal transduction role in myocardial regeneration. Using the tcf21: nucEGFP transgenic line to observe the behavior of epicardial cells, the results showed in both control and mvp-/- animals that the epicardial cells around the ventricle were stimulated by injury and proliferated, and 7 days after injury (dpa) Merge into the underlying myocardial wall. To determine whether the loss of mvp affects the regenerative capacity of the zebrafish heart, we examined cardiomyocyte proliferation, scar tissue removal and muscle recovery. In all cases, no significant differences were observed between mvp-/- and wild-type animals. These results indicate that the heart regeneration ability of mvp–/−zebrafish has not changed.

　　It is reported that the inhibitory effect of morpholino on MVP in zebrafish leads to a decrease in axon regeneration after spinal cord injury. In order to investigate whether mvp affects the regeneration of the central nervous system, axon regeneration and motor recovery in the zebrafish spinal cord transection model were studied. The regeneration of the central nervous system of zebrafish occurs through new neurogenesis after the activation and proliferation of neural progenitor cells. To test whether MVP is required for regenerative cell proliferation, we quantified the cell proliferation of damaged spinal cord, and no significant difference was observed between mvp-/- and wild-type animals. Using acetylated α-tubulin immunohistochemical methods, regenerating axons were observed in transected sites in most wild-type and mvp-/- samples 30 days after injury (dpi). Retrograde tracking of axonal projections confirmed the reconnection of the head and tail stumps of the two types of spinal cords. The motor activity of the fish is evaluated as an indicator of functional recovery. The data showed that the wild-type control group and mvp−/− fish recovered their pre-injury swimming performance at 30 dpi. The above results indicate that the lack of MVP has no significant effect on the regeneration of the zebrafish spinal cord.

　　Then, the key process of mvp-/-zebrafish fin regeneration was studied. In mvp-/- animals, the cell proliferation in the interstitial area is comparable to that of the wild type. In order to further examine the fin regeneration ability of mvp-/-fish, the tail fins of wild-type and mvp-/-fish were truncated, and the growth of new fin tissue was continuously monitored for 14 days. The regeneration rate of mvp-/-fish is similar to that of wild type.

　　mvp knockout zebrafish cell death and transcriptome changes: Just like humans, damage can cause rapid local tissue necrosis and trigger zebrafish cell apoptosis. More and more evidences show that MVP participates in cell survival by regulating the expression of apoptosis-regulating genes (such as Bcl-2). Therefore, we detected apoptosis in the damaged heart and spinal cord, and TUNEL analysis showed that cell death in these two tissues was significantly enhanced in mvp–/−fish. The previous results showed that the regenerative ability was preserved in mvp knockout zebrafish. The transcriptome changes in wild-type and mvp-/- animal regenerated hearts were analyzed by RNA-seq to check whether the regeneration program was changed. PCA analysis shows that the distribution of the transcriptome is closely related to the state of heart regeneration, which indicates that the response of mvp-/- animals to regeneration signals is similar to that of wild-type. Zebrafish heart regeneration is a highly dynamic process involving multiple signals and factors, forming a complex regeneration process. The expression of regeneration-related genes was checked, and it was found that most of them showed similar transactivation levels in mvp-/- animals as wild-type. In summary, these data indicate that MVP plays an anti-apoptotic effect in the early stage of the injury response, and the loss of MVP has no obvious effect on the regeneration process of the heart.

　　Discussion: In this study, the MVP gene knockout zebrafish strain was successfully constructed using CRISPR/Cas9 technology. The entire coding region of MVP (about 22kb) was knocked out, and mvp mRNA was not detected in knockout animals, so the allele was a true null allele. Like mammals, the fornix does not play an important role in the development of bones and the cellular processes in normal environments. The data further indicates that the vault is involved in the mechanisms required for cell survival under injury-induced cell stress. This result is consistent with the observation that the mvp of the three tissues analyzed is transactivated in the acute phase of the injury response. Extensive cell death was found in mvp knockout zebrafish, and no long-term effects on regeneration were detected. A possible explanation for this observation is that the extraordinary regeneration ability of zebrafish has fully supplemented the excessive cell loss. It is interesting to study the effect of mvp loss on organ regeneration/tissue repair in animals with limited regenerative capacity. Since MVP knockout mice are feasible, this type of experiment is feasible and can provide a reference. In addition to MVP, the fornix also contains two other proteins, TEP1 and VPARP, and a non-coding RNA (vRNA). According to reports, in zebrafish, MVP acts as a negative regulator of IFN production by limiting the activation of TBK1. Transcriptome data showed that the inflammation intensity of injured mvp knockout fish did not change significantly. A more systematic analysis is needed to clarify the role of mvp on the immune response in various situations.