A method for constructing a cofilin-1 transgenic model and use thereof

Abstract

This invention provides a transgenic animal established by expressing filament protein using the Cre-loxP conditional expression method, which allows for observation and evaluation of the pathophysiological response when the protein is overexpressed in vivo.

Description

A method for constructing a fibronectin gene transfer model and its application The present invention relates to an application for preparing a model for transfecting a fibronectin gene, and particularly to an application for preparing a multicellular organism model for transfecting a fibronectin gene. Aging is a significant risk factor for a variety of human diseases, including cognitive impairment, cancer, arthritis, vision loss, osteoporosis, diabetes, cardiovascular disease, and stroke. In addition to the normal synapse loss that occurs during natural aging, synapse loss is a common early pathological condition in many neurodegenerative diseases and is most closely associated with the neuronal and cognitive impairments associated with these diseases. Therefore, aging remains the primary risk factor for dementia-related neurodegenerative diseases such as Alzheimer's disease (AD). Aging affects all tissues and functions of the body, including the central nervous system, and the decline in functions such as cognition and motor activity can seriously affect quality of life. On the other hand, Cofilin-1, which is expressed by mammalian cells, is 19 kDa (Chen H et al., (2000) Trends Biochem Sci. 25(1): 19-23). ​​The cofilin family consists of non-muscle cofilin-1 (n-cofilin) ​​(also known as type I cofilin), muscle cofilin-2 (m-cofilin) ​​and actin depolymerizing factor (ADF) (Ono S et al., (2007) Int Rev Cytol. 258: 1-82; Poukkula M et al. (2011) Int Rev Cytol. 68: 471-90). Among them, non-muscle fibronectin is an essential protein widely expressed in various tissues and organs. In addition, fibronectin is a microfilament-binding protein whose main function is to regulate the depolymerization of microfilaments, which further affects the shape, migration, division and other functions of cells. Previous studies have found that when cells over-express fibronectin, the cell shape will become larger and promote cell aging. For example, in the human brain, both ADF and Cofilin-1 are expressed, and the expression level of Cofilin-1 is 10 times higher than that of ADF (Bellenchi GC et al., (2007) Genes Dev. 21:2347-57). Cofilin maintains synaptic plasticity by regulating the structure of dendritic spines in neurons, which is associated with memory and learning. However, dysregulation of ADF / cofilin activity is associated with brain aging and cognitive decline. Under various pathological conditions, this may lead to ischemic and hemorrhagic stroke, neurodegenerative diseases (such as Alzheimer's and Parkinson's), and genetic disorders. In addition, existing genetic technology model animals can be roughly divided into gene knockout technology and gene transfer technology. However, existing studies on animal models of aging have mostly used cofilin gene knockout techniques to explore the role of cofilin in organisms. For example, it is currently known that gene knockout in yeast, mice, or mammalian cells can cause cell death, including embryonic lethality. Overexpression of cofilin has only been shown to inhibit cell cycle progression, aging, DNA repair, cell migration, and tumor cell metastasis at the in vitro cellular level, but has not significantly promoted cell death. Functional studies of cofilin overexpression are mostly limited to biochemical and cellular biological levels, and there is still no technology to transfer the gene into multicellular organisms and enable its expression for more direct biological observation, which would serve as a basis for biomedical research and disease treatment. Therefore, the development of model animals for inducible expression of transgenic cofilin technology and its application in anti-aging drug screening platforms are urgent issues that need to be addressed in this field. In view of the above, the object of the present invention is to develop a novel method for inducing the expression of cofilin gene in multicellular organisms. Furthermore, the model animals of transgenic technology can be divided into the first generation of constitutive Cre, the Cre- Model animals of the loxP recombinase system (Cre-ERT) and the second generation of inducible Cre- Model animal of the loxP recombinase system (Cre-ERT2). Cre-ERT2 is a fusion protein of Cre and Estrogen receptor (ER). In the absence of Tamoxifen (TAM), Cre and ER will interact with heat shock protein 90 (HSP90) in the cytoplasm. If Tamoxifen (TAM) is added to the cells, HSP90 will be removed, and Tamoxifen (TAM) will combine with Cre-ER to form a complex and enter the cell nucleus. At this time, Cre will find the cell with The loxP-containing gene Y removes the Y gene, allowing the gene following the Y gene to be expressed in large quantities. Therefore, Cre-ERT2 is called the inducible Cre-ERT system (Kim H et al., (2018) Lab Anim Res. 34: 147-59). Furthermore, the present invention has experimentally verified that Cre-ERT can externally control the activity of the recombinase compared to the constitutive Cre, thereby improving the accuracy of gene manipulation, thereby verifying the second generation of inducible Cre- The sensitivity and specificity of the loxP recombinase system (Cre-ERT2) are better than those of the first generation Cre- The loxP recombinase system (Cre-ERT) is preferred. On the other hand, another purpose of the present invention is to use gene transfer technology to enable the expression of cofilin in multicellular organisms in large quantities, and to explore whether the expression of cofilin in multicellular organisms in large quantities will accelerate the aging of tissues and organs of organisms. loxP recombinase system (Cre-ERT2) to induce Cofilin-1 ( After Cfl1 is expressed in large quantities in model organisms, the aging phenomena of the aforementioned multicellular organisms are observed, for example, the aging phenomena of the animal nervous system. Furthermore, the present invention further applies the aforementioned developed method to a screening platform for anti-aging drugs. The following embodiments are used in conjunction with the accompanying drawings to illustrate the present invention in detail. The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. Figure 1 is a schematic diagram of the breeding process of transgenic mice according to one embodiment of the present invention. Genotyping of mice born from mating of loxP-emGFP-loxP-Cfl1 transgenic mice (second generation, F0). Figures 2A-2C show the differences between wild-type mice (WT) that do not express green fluorescent protein and do not carry exogenous cofilin (Figure 2A) and mice that express the fluorescent gene but also carry exogenous cofilin. loxP-emGFP-loxP-Cfl1 mice, but will not be expressed (Figure 2B). Figure 2C is a genotyping gel electrophoresis of Figure 2A and Figure 2B, used to compare wild-type (WT) mice (Lane 1) and Differences in loxP-emGFP-loxP-Cfl1 mice (Lane 2) carrying exogenous cofilin fragments. Figure 3 refers to Genotyping results for offspring of loxP-Cfl1 mice mated with wild-type (WT) or UBC-Cre mice: Cells #34, #36, and #37 are fluorescent, seemingly successful, but this is not the case. This is because subsequent Cre genotyping was unsuccessful in these mice. Figure 4 is a schematic diagram of the breeding process of transgenic mice according to another embodiment of the present invention. When breeding loxP-emGFP-loxP-Cfl1 males or females (first parent, F0) with Cre-ERT2 males or females (second parent, F0), Cofilin-1 will not be expressed in large quantities because emGFP is followed by a stop-cassette. Cre-ERT2(+) / Cfl1(+ ) mice (first generation, F1), After loxP-emGFP is pop-out through tamoxifen (TAM) treatment (as shown in the figure "F1+TAM"), Cofilin-1 can be expressed in large quantities. 〔Figure 5〕$47~$54 are the genotyping results of the pups born from Cre-ERT2 (red group number 5 (red 5), ♂) and WT (blue group number 4 (blue 4), ♀). $47, $49, $50, $52 and $53 are Cre-ERT2(+). Cre: 560bp. M: 100bp Marker. P: Cre-ERT2 positive control. 〔Figure 6〕shows #30~#37 The genotyping results of the offspring of loxP-Cfl1#51♂ and WT♀ (blue group number 1 (blue1)) are #30, #32, #34 and #35. loxp-Cfl1(+). M: 100bp Marker. P: loxP-emGFP-loxp-Cfl1( loxp-Cfl1)positive control. loxP-Cfl1: 423bp, WT: 320bp. 〔Figure 7〕shows *55~*62 The genotyping results of the offspring mice born from breeding loxP-Cfl1#54♂ and Cre-ERT2 (red group number 4 (red 4),♀) are *55, *57, *58, and *61. Cre-ERT2(+) / Cfl1(+). M: 100bp Marker. P: Cre-ERT2 / Cfl1 positive control(*28). [Figure 8] *74~*83 The genotyping results of the offspring mice born from breeding loxP-emGFP-loxP-Cfl1 (loxP-Cfl1)#54 (♂) and Cre-ERT2 (red group number 3 (red 3), ♀) are *74, *76, *78, *79, *81 and *82. Cre-ERT2(+) / Cfl1(+). M: 100bp Marker, P: Cre-ERT2 / Cfl1 positive control. 〔Figure 9〕shows *28 is The offspring of loxP-emGFP-loxP-Cfl1 (loxP-Cfl1)#54 (♂) and Cre-ERT2 (red group number 4 (red 4), ♀). *42 is Pups born from loxP-emGFP-loxP-Cfl1 (loxP-Cfl1)#52 (♂) and Cre-ERT2 (red group number 3 (red 3), ♀). *28 and *42 are mice with cofilin-1 inserted. M: 100bp marker. P1: positive control, plasmid Cfl1 DNA diluted 10X. P2: positive control, plasmid Cfl1 DNA diluted 20X. Plasmid Cfl1 DNA concentration: 781 ng / μl. Cofilin-1: 500 bp. Figure 10 Cre-ERT2(+) / Cfl1(+) mice During the loxP-emGFP pop-out period (during tamoxifen (TAM) feeding), the status of tamoxifen (TAM) consumption and changes in body weight were shown. In the figure, "(1)" indicates that *42 was fed a tamoxifen (TAM) diet and monitored by a non-invasive 3D intravital molecular imaging system (IVIS); "(2)" indicates that *42 had lost approximately 20% of its body weight on the fourth day of the tamoxifen (TAM) diet, so some normal diet was mixed in. In this experiment, when mice were first switched to tamoxifen (TAM) feed, they did not eat the tamoxifen (TAM) feed immediately, so their weight would decrease. After a transition period, the mice would start eating the tamoxifen (TAM) feed and their weight would recover. "(3)" indicates the time when the mice started eating tamoxifen (TAM); "(4)" indicates the 7th day after they stopped eating the tamoxifen (TAM) feed, when their weight recovered significantly; "(5)" indicates the 69th day after they stopped eating the tamoxifen (TAM) feed, when their weight was the same as that of the wild type (WT). In addition, " ” indicates the time when mice started to eat tamoxifen (TAM) diet; "" indicates that the ears and tails of mice were removed for genotyping and IVIS analysis on the 21st day, and the tamoxifen (TAM) diet was stopped. Figure 11 shows Cre-ERT2(+) / Difference in photon counts in Cfl1(+) mice before (A) and 21 days after (B) feeding with tamoxifen (TAM) diet. Figure 12 shows that mice On days 21 and 35 after loxP-emGFP pop-out (i.e., feeding with tamoxifen (TAM)), After loxP-emGFP pop-out, a 247bp DNA fragment can be seen. M: 100bp marker. B: Genomic DNA before loxP-emGFP pop-out. Figure 13 Cre-ERT2(+) / Cfl1(+) mice fed with tamoxifen (TAM) diet Cofilin-1 RNA expression level.**: p<0.01. [Figure 14] shows (A) HE staining of tissue sections of wild-type (WT) mouse ears; (B) IHC staining of cofilin-1 in tissue sections of wild-type (WT) mouse ears; (C) Cre-ERT2(+) / HE staining of tissue sections of Cfl1(+) mouse ears; (D) Cre-ERT2(+) / Cofilin-1 IHC staining of a tissue section from the ear of a Cfl1(+) mouse. Magnification: 20X. Scale: 50 μm. Figure 15 shows the photographic monitoring of old wild-type mice (WT old mouse), wild-type (WT) and Cre-ERT2(+) / The difference in activity frequency between Cfl1(+) mice. The darker lines represent the activity time recorded by the camera; the more frequent the activity, the denser the lines. Although preferred embodiments of the present invention have been shown and described herein, these embodiments are provided by way of example only and are not intended to limit the scope of the present invention in other ways. Various alternatives to the described embodiments of the present invention may be employed in practicing the present invention. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In one embodiment of the present invention, a method for constructing a multicellular organism transfected with a fibronectin gene comprises the following steps: Step 1: (1) The first parent has loxP recombinant DNA fragment The first DNA plasmid constructed by Actb gene, The loxP recombinant DNA fragment contains a green fluorescent protein (GFP) gene and a Neomycin resistant (NeoR) fragment. The Actb gene line contains the fibronectin gene, The loxP recombinant DNA fragment was ligated to the front end of the Actb gene; (2) The second parent has a second DNA plasmid constructed by the Cre recombinant protein gene or the Cre-ERT2 fusion protein gene; Step 2: The first parent (1 st F0) and the second parent (2 nd F0) is mated to obtain the first offspring (F1). In other words, if the parent's genotype is loxP-emGFP-loxP-Cfl1( loxP-Cfl1) and Cre-ERT2, the genotype of the first progeny may include Cre-ERT2(+) / Cfl1(+ )genotype, Cre-ERT2(+) / Cfl1(- )genotype, Cre-ERT2(-) / Cfl1(+ ) genotype and Cre-ERT2(-) / Cfl1(- ) genotype, etc. In another embodiment of the present invention, the cofilin gene can be selected from, but not limited to, non-muscle-type cofilin genes or muscle-type cofilin genes. In yet another embodiment of the present invention, Cre-ERT2 is an "inducible promoter". The aforementioned "inducible promoter" requires tamoxifen (TAM) to induce the expression of Cofilin-1. Once induced, Cofilin-1 will be continuously expressed. On the other hand, constitutive Cre does not require any induction, as long as the offspring have both constitutive Cre and loxP-emGFP-loxP-Cfl1 gene, constitutive Cre will automatically loxP-emGFP pop-out allows Cofilin-1 to be continuously expressed in large quantities. The model described in one embodiment of the present invention can be an individual, and the aforementioned individual can be a multicellular organism, such as a human or a non-human animal, wherein the non-human animal is, for example, a cat, dog, rabbit, cow, horse, pig, sheep, goat, monkey, guinea pig, gerbil, rat, mouse, zebrafish, nematode, fruit fly, etc. In one embodiment of the present invention, the fibronectin gene was firstly transfected and expressed in mice. By constructing a "STOP-n-GO" gene expression framework, the fibronectin fused with a 3X peptide tag (Flag) was implanted into a DNA plasmid, which covers the mouse Actb gene, and the aforementioned silk protein gene is selected and cloned in it. And the front end of the gene is inserted into a segment consisting of loxP recombinant DNA fragments, which contain the green fluorescent protein gene (GFP) and Neomycin resistant (NeoR) fragments, can be used for gene transfer in various species, including small mammals and through Cre- LoxP recombination technology controls the in vivo expression of cofilin. The Cre recombinase gene is carried by a separate plastid. Expression of the gene can be achieved through a constitutive or tissue-specific driver, or by combining a Cre-ERT2 fusion gene with tamoxifen (TAM) to induce the protein into the cell nucleus for recombination. All of these approaches can achieve cofilin gene expression in multicellular organisms. In short, this technology allows for the observation of GFP fluorescence to manipulate and evaluate the phenomenon and biological effects of induced cofilin gene expression. In other words, the present invention verifies that fibronectin can be systemically expressed in multicellular living organisms, and can also encompass other methods for establishing transgenic expression of the protein, thereby providing a research platform for cross-disciplinary biomedical research. The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for illustrative purposes only, and the disclosure contained herein should in no way be interpreted as being limited to these examples, but rather should include all results that are significantly modified by the disclosure of the present invention. Each treatment condition was repeated at least three times in the experiment. For in vivo experimental determinations, the number of mice used in each group is indicated. Data are expressed as mean ± SD. Data collected from at least three independent experiments were statistically analyzed, and statistical results are shown as mean ± SEM. For representative images, at least three independent experiments showed similar results. Comparisons between groups were analyzed using Student's t-test or one-way ANOVA. The results of the statistical analysis were considered significant when p < 0.05. Example Example 1: Extraction of Genomic DNA Collect tissues to be analyzed, including: (a) Clip the toes of the pups on day 10 after birth, place the little toes in an eppendorf tube, and add 200 μl of DirectPCR lysis reagent if there are two little toes, or 100 μl of DirectPCR lysis reagent if there is only one little toe. (b) For mice with loxP-emGFP pop-out (after TAM treatment), anesthetize with 1-3% isoflurane and remove a small ear fragment (approximately 6 mg). Add 100 μl of DirectPCR lysis reagent. Add 2 μl of Proteinase K (stock: 20 mg / ml) per 100 μl of DirectPCR lysis reagent, for a final working concentration of 0.4 mg / ml. Vortex and mix thoroughly, then heat in a 55°C water bath overnight. Inactivate the fragment by heating in an 85°C water bath for 45 minutes. Centrifuge the sample at 8000 rpm for 1-2 minutes, remove the supernatant, and transfer it to a new Eppendorf tube. Store the genomic DNA at -20°C until ready for use. Example 2: Polymerase Chain Reaction (PCR) The primer names and sequences used in the polymerase chain reaction of the present invention are shown in Table 1. In other words, in one embodiment of the present invention, the transgenic animal genotype identification primer sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 8. Table 1. Primer names and sequences (F: forward primer; R: reverse primer) After calculating the required amount of primers, you can prepare the primers. Dilute 10μM forward primer and 10μM reverse primer 10-fold in RNase-free water at a ratio of 1:9. Then mix the forward primer and reverse primer at a ratio of 1:1. The polymerase chain reaction solution can be prepared according to Table 2. Table 2. Polymerase chain reaction solution The reaction conditions of polymerase chain reaction in polymerase chain reaction instrument are shown in Table 3. The reaction temperature for step 3 (annealing temperature) of the Actb (pup-out) primer was 57°C, and that of the other primers was 55°C. Table 3. Reaction conditions of polymerase chain reaction instrument Agarose gel electrophoresis was used to analyze the size of the polymerase chain reaction product. Table 4. PCR product size Example 3: Breeding of transgenic mice expressing persistent induction Please refer to Figures 1, 2A to 2C, and 3. One embodiment of the present invention uses transgenic mice (UBC-Cre) that continuously express the Cre recombinase gene and The process and genotyping results of mice born from the mating of loxP-emGFP-loxP-Cfl1 transgenic mice. Please also refer to Figures 2A-2C, where the genotyping in Figure 2C was obtained by PCR. Lane 1 in Figures 2A and 2C represents wild-type mice that do not express green fluorescent protein and do not harbor exogenous cofilin. Lane 2 in Figures 2B and 2C represents mice that express the fluorescent gene but also harbor exogenous cofilin (a higher molecular weight PCR fragment), but it is not expressed. Green fluorescence can be observed in the eye in the image shown in Figure 2B. This poses the problem of false positives. by loxp-Cfl and wild type (WT) were bred and gave birth to offspring with The mouse samples of loxp-Cfl (#32, #23) and those without transgenic gene (#22) were used as comparison. The results of gene identification of mice after loxp-Cfl mating, including those with fluorescence (#34, #36, #37) and those without fluorescence (#35), respectively, for Cre gene and After PCR of the loxp-Cfl gene, the fluorescent results were the same as #32 and #23, while the fluorescent #35 was the same as #22, indicating that it was the wild type (WT) without the transgenic gene (as shown in Figure 3). In other words, mice that continuously express Cre are used for breeding If the offspring of loxp-Cfl continue to show fluorescence, it means that the STOP-n-GO sequence has not been removed. Cfl was also not expressed. However, mice that did not express fluorescent protein also did not The expression signal of Cfl and most importantly the Cre gene were not expressed in these newborn mice, indicating that the transgenic mice that continue to express Cre are The results of mating loxp-Cfl transgenic mice and giving birth to mice are all failures. It is also possible that the embryos express a large number of Cfl will affect the development of the embryo and lead to failure in successful delivery. Furthermore, the use of transgenic mice that continuously express the Cre recombinase gene (UBC-Cre) and After breeding, loxP-emGFP-loxP-Cfl1 transgenic mice often lead to dystocia and death of pregnant female mice. Transgenic mice with Cfl are not easy. Example 4: Breeding of transgenic mice expressing non-sustained induced expression C57BL / 6 mouse background is divided into: (a) Cre-ERT2 breeding: Cre-ERT2 and wild-type (WT) males and females are caged together; (b) loxP-emGFP-loxP-Cfl1( loxP-Cfl1) seed preservation: LoxP-Cfl1 and wild-type (WT) males and females were caged together; (c) Cre-ERT / Cfl1 breeding: will carry Cre-ERT2 and The loxP-Cfll breeding male and breeding female are put into the same cage, and the cage date and breeding male or breeding female number are recorded. The breeding female can be observed every other day to see if there is a plug. If no plug is observed during the cage period, it can be observed about two weeks after the cage is put into the same cage to see if there is any sign of pregnancy (for example, the breeding female's belly becomes bigger). The gestation period of the female mouse is three weeks. At this time, the breeding male can be removed and nesting material can be put in to allow the female mouse to build a nest and give birth, and pay attention to the time of delivery. The female mouse and the pups should not be disturbed for the first three days after giving birth, otherwise the female mouse will eat the mice. On the 10th day after the birth of the pups, the toes need to be clipped, numbered, and genotyping analysis should be performed to see if they carry the target genes. The symbol code for Cre-ERT2 mice is $, The symbol code of loxP-Cfl1 mouse is #, The symbol for Cre-ERT2 / Cfl1 mice is *. Genotyping confirmed Cre-ERT2(+) / Cfl1(+ ) mice, the eyes of the mice can be seen with green fluorescence. When the mice are about 8 to 10 weeks old, they need to be fed with Tamoxifen (TAM) feed. Deletion of loxP-emGFP resulted in the abundant expression of Cofilin-1 ( FIG4 ). Please refer to Figure 5, which shows the genotyping results of Cre-ERT2 seedlings. In the figure, 560 bp of DNA is found, which is the carrier of Cre-ERT2(+) mice. The Cre-ERT2(+) reproduction rate was approximately 48%, with 16% of males and 32% of females. The pup culling rate was 26%, and the pup mortality rate was 26%. Please refer to Figure 6 for The genotyping results of loxP-Cfl1 seed conservation, loxP-Cfl1(+) has two DNA sizes, 423 bp ( loxP-Cfl1) and 320bp (WT). The loxP-Cfl1(+) reproduction rate was 36%, with 18% for males and 18% for females. The pup culling rate was 26%, and the pup mortality rate was 24%. The genotyping of Cre-ERT2 / Cfl1 breeding mice is shown in Figures 7 and 8. Four different genotypes were observed in the offspring of Cre-ERT2 / Cfl1 mice: (1) WT: *56 and *60, DNA size was 320 bp (Figure 7); (2) Cre-ERT2: *59, *62, *80, and *83, DNA size was 560 bp and 320 bp (Figures 7 and 8); (3) loxP-Cfl1: *75 and *77, DNA sizes are 423 bp and 320 bp; (4) Cre-ERT2(+ ) / Cfl1(+ ) In Figure 8 , three bands can be seen, with DNA sizes of 560 bp, 423 bp, and 320 bp, respectively. Cre-ERT2(+) / The Cfl1(+) reproduction rate was 26%, with 14% for males and 12% for females. Furthermore, the pup culling rate was 51% and the pup mortality rate was 24%. The PCR results in Figure 9 show Cre-ERT2(+) / Cofilin-1 is indeed incorporated into the DNA of Cfl1(+) mice. Example 5: Transgenic mice loxP-emGFP can be knocked out (pop-out) by feeding tamoxifen (TAM) and body weight monitoring Genotyping confirmed with Before feeding Cre-ERT2 / Cfl1 mice a tamoxifen-containing diet (400 ppm), measure their body weight. During the tamoxifen diet, measure both body weight and the amount of tamoxifen diet three times a week to monitor the mice's food intake and the amount of tamoxifen consumed. If the mice lose 20%-25% of their body weight, mix one-third of their normal diet with the tamoxifen diet to restore their weight and improve their acceptance of the tamoxifen diet. In other words, please refer to Figure 10. Cre-ERT2(+) / The changes in body weight of Cfl1(+) mice during the period of feeding with Tamoxifen (TAM) diet were compared with the changes in body weight of Cfl1(+) mice during the period of feeding with Tamoxifen (TAM) diet. Cre-ERT2(+) / Cfl1(+) mice lost 20% of their body weight and were fed a normal diet containing one-third of their body weight. Cre-ERT2(+) / Cfl1(+) mice regained weight and began to consume tamoxifen (TAM) feed, consuming a total of 37.4 g of TAM feed (containing ~15 g of tamoxifen (TAM)) over 21 days. loxP-emGFP pop-out period Cre-ERT2(+) / The body weight of Cfl1(+) mice was maintained at 16-17 g. On the 69th day after stopping the tamoxifen (TAM) diet, Cre-ERT2(+) / The body weight of Cfl1(+) mice increased to the same level as that of WT mice. Example 6 Cre-ERT2(+) / Cfl1(+) mice Optical image analysis of loxP-emGFP knockout by tamoxifen (TAM) administration Mice were anesthetized with 3% isoflurane and placed in an imaging box. 1-2% isoflurane was used to maintain the mice under anesthesia. Fluorescence intensity was analyzed using the Biospace Lab PhotonIMAGER Optima optical imaging system. The eGFP imaging mode was selected, with an excitation light of 487 nm and an emission light of 522 nm, and an exposure time of approximately 5 seconds. Regions of interest (ROIs) were circled using Biospace Lab M3 Vision software, and photon flux was analyzed throughout the mouse body for comparison. Differences before and after loxP-emGFP pop-out. Figure 11 and Table 5 show Cre-ERT2(+) / The difference in the number of whole body photons in Cfl1(+) mice after feeding TAM diet. The results in Figure 11 and Table 5 show that after feeding TAM diet, Cre-ERT2(+) / The number of photons in the whole body of Cfl1(+) mice is reduced, indicating loxP-emGFP was deleted by tamoxifen (TAM). The PCR results (Figure 12) also showed that Actb and the second The primer designed with loxP sequence was used to detect the effect of TAM on feeding for 21 and 35 days. Cre-ERT2(+) / Cfl1(+) mouse genomic DNA, After loxP-emGFP was pop-out, a 247 bp DNA was clearly visible. Table 5 is After loxP-emGFP pop-out, Cre-ERT2(+ ) / Cfl1(+ ) Changes in photon counts in mice In one embodiment of the present invention, when Cre-ERT2(+) / When Cfl1(+) mice ingested approximately 15 g of tamoxifen (TAM), the photon count of emGFP decreased from 9.27E+10~8.31E+10 to 6.56E+10~5.63E+10 (as shown in Table 5). Example 7 Cre-ERT2(+) / Cfl1(+) mice Cofilin-1 RNA expression after loxP-emGFP pop-out To extract RNA from model animal ear tissue, sterile scissors were used to harvest approximately 6-8 mg of mouse ear tissue. The tissue was placed in a microcentrifuge tube and stored in liquid nitrogen until RNA extraction, at which point the cryovial was removed. The ear tissue in the microcentrifuge tube was ground on dry ice using an electric grinder. TRIzol reagent was added to each microcentrifuge tube, and chloroform was added according to the protocol, followed by vortexing, centrifugation, and other experimental steps. The supernatant (aqueous phase) was transferred to a fresh microcentrifuge tube. Add 250 μl of isopropanol to each microcentrifuge tube, gently mix by inverting, and allow to stand for 10 minutes. Remove the sample after the reaction and place it on a thin-film column for RNA purification. Centrifuge, add 75% ethanol, centrifuge again, and dry. Add 30-50 μl of RNase-free water, allow to stand for 3 minutes, and centrifuge at 16,000 g for 1 minute at 4°C. Quantify RNA; the amount of RNA dissolved in 50 μl of RNase-free water is approximately 30-50 ng / μl (approximately 6-8 mg of ear tissue). In addition, 100-150 ng of RNA was reverse transcribed into cDNA using the ToolsQuant II Fast RT Kit (KRT-BA06; BioTools, Taipei, Taiwan) and its manual. Finally, cDNA was quantified to yield approximately 1100-1200 ng / μl. For the primer names and sequences of quantitative polymerase chain reaction (qPCR), please refer to Table 6 below. Table 6. Primer names and sequences (F: forward primer; R: reverse primer) The concentration of the primers required for qPCR is 6 μM. The quantitative polymerase chain reaction solution can be prepared according to Table 7. Table 7. Quantitative polymerase chain reaction solution The samples prepared as described above were then placed in a real-time polymerase chain reaction (PCR) instrument (StepOnePlus) for qPCR analysis under the following conditions. Table 7. Reaction conditions of quantitative polymerase chain reaction instrument Cre-ERT2(+) / After Cfl1(+) mice were fed with tamoxifen (TAM) diet, RNA from ear tissues was collected for qPCR analysis. The qPCR analysis results showed that Cre-ERT2(+) / After Cfl1(+) mice were fed a tamoxifen (TAM) diet, the expression level of cofilin-1 was 4.9 times that of wild-type (WT) mice ( FIG13 ). Example 8 Cre-ERT2(+) / Cfl1(+) mice Immunohistochemistry (IHC) was used to observe the protein expression of Cofilin-1 after loxP-emGFP pop-out. After paraffin embedding, the tissue was sectioned and heated in a 60°C oven for 1 hour to dewax. The dewaxed tissue was washed twice with Xylene for 5 minutes, then twice with 95% and 75% alcohol for 3 minutes each, and then twice with PBST for 5 minutes. The sections were placed in 10 mM citric acid buffer (containing 0.1% Tween-20, pH 6) and heated at 121°C for 4 minutes for epitope retrieval. The sections were then allowed to cool at room temperature for approximately 20 minutes. After cooling, the sections were soaked in PBST for 5 minutes, removed, and excess water was removed from the slides using lens tissue. The tissue was circled with a pen, and Dual Endogenous Enzyme Blocking Solution was applied to the sections. The sections were incubated in the dark for 5 minutes, washed with PBST for 5 minutes, and excess water was removed from the slides using lens tissue. Goat serum was then applied to the sections and incubated in the dark for 30 minutes. Wash with PBST for 5 minutes, remove excess water from the slide with lens tissue, and then drop the primary antibody (Cofilin-1:PBST=1:100) on the tissue and incubate at 4°C overnight. Wash with PBST for 5 minutes, add 100μl of Labelled polymer-HRP secondary antibody and incubate for 20 minutes. Wash twice with PBST for 5 minutes, add 200μl of substrate working solution and incubate in the dark for 30 seconds. Wash with PBST for 5 minutes, add 100μl of Mayer's Hematoxylin to the tissue slices and stain for 30-60 seconds. Use ddH 2O to rinse the sliced ​​tissue. The sliced ​​tissue needs to be reverse dewaxed and washed with ddH After washing with 2O for 3 minutes twice, washing with 75% and 95% alcohol for 3 minutes twice, respectively, and washing with Xylene for 5 minutes twice, excess water on the slide was removed with lens paper and then mounted. Please refer to Figure 14. Cre-ERT2(+) / Cfl1(+) mice were fed with tamoxifen (TAM) diet and Cre-ERT2(+) / Results of IHC analysis of ear tissues from Cfl1(+) mice. Cre-ERT2(+) / The difference in the protein expression of Cofilin-1 between Cfl1(+) mice and wild-type (WT) mice is shown in Figure 14 by the results of immunohistochemical staining (IHC). Cre-ERT2(+) / Cofilin-1 was expressed in greater amounts in the ear muscles of Cfl1(+) mice than in WT mice. In summary, in one embodiment of the present invention, a Cre-ERT2 animals and a The loxP-emGFP-loxP-Cfl1 animals were mated to obtain an F1 generation animal; wherein the genotype of the F1 generation animal was identified as Cre-ERT2(+) / Cfl1(+ ) genotype animals. Cre-ERT2(+) / Cfl1(+) genotype animals were fed tamoxifen (TAM) diet. Deletion of loxP-emGFP resulted in the expression of a large amount of Cofilin-1 in the animal. Example 9, old wild-type mice (WT old mouse), wild-type mice (WT mouse) and Cre-ERT2(+) / Differences in activity frequency in Cfl1(+) mice In one embodiment of the present invention, the function of the fibronectin transgenic gene in small animals is observed by observing the frequency of animal activity. The activity frequency of mice was recorded for 24 hours using a camera, and the activity of wild-type (WT) mice, Activity differences between Cre-ERT2 / Cfl1 mice and aged wild-type mice (WT old mice). Figure 15 shows 24-hour video monitoring of activity frequency. During the active period (20:00-08:00), activity frequency of 78-week-old wild-type mice (78W) was lower than that of 19- and 21-week-old mice (19W and 21W), respectively, for both male and female mice. Cre-ERT2(+) / The activity frequency of Cfl1(+) mice during the active period is less continuous than that of wild-type (WT) mice of the same age. The activity pattern of wild-type mice of the same age during the active period is continuous. During the sleep period (08:00-20:00), Cre-ERT2(+) / The activity frequency of Cfl1(+) mice was slightly higher than that of wild-type mice of the same age. In summary, the present invention shows that Cre-ERT2(+) / When Cfl1(+) mice were fed 15g of tamoxifen (TAM), the photon count of emGFP decreased from 9.27E+10~8.31E+10 to 6.56E+10~5.63E+10 (Table 5), which was also confirmed by the results of genotyping, qPCR and IHC (Figures 12-14). Cre-ERT2(+) / When Cfl1(+) mice were fed with this dose of tamoxifen (TAM), LoxP-emGFP can be popped out to increase the expression of Cofilin-1. The effective dose of Tamoxifen (TAM) for Cre recombinase activation is 15g. When mice are fed with Tamoxifen (TAM) diet, their body weight is maintained at 16-17g (Figure 10). In addition, the present invention also shows The reproduction rate of Cre-ERT2(+) was 48%, The propagation rate of loxP-Cfl1(+) was 36%, Cre-ERT2(+) / The Cfl1(+) reproduction rate was 26%, and Cre-ERT2(+) / The elimination rate of Cfl1(+) can reach 51%; Cre-ERT2(+) / Cfl1(+) is less likely to reproduce. Cre recombinase needs to be activated by tamoxifen (TAM). Cre-ERT2(+) / When Cfl1(+) mice are first switched to a tamoxifen (TAM) diet, they have an adaptation period of about 6 days, and their body weight is maintained at 16-17g during the tamoxifen (TAM) administration period. When tamoxifen (TAM) is stopped, Cre-ERT2(+) / The body weight of Cfl1(+) mice gradually increases to the same level as that of wild-type (WT) mice. Cre-ERT2(+) / The activity frequency of Cfl1(+) mice during the active period (20:00-08:00) is similar to that of older mice, both of which are in a more discontinuous state, while the activity frequency of wild-type (WT) mice is more continuous, indicating that a large amount of Cofilin-1 is expressed. Cre-ERT2(+) / The brains of Cfl1(+) mice have already begun to undergo changes, suggesting that they are heading towards a neurodegenerative disease process. That is, in one embodiment of the present invention, multicellular organisms transfected with the fibronectin gene, such as mice, zebrafish, nematodes, and fruit flies, can be used to screen, prepare, detect, and / or treat anti-aging drugs and serve as an anti-aging drug screening platform. The foregoing detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described; many variations will be apparent to those skilled in the art and are intended to be included in the invention as defined by the claims. TW202415771A_111138901_SEQL.xml

Claims

1. An application that can induce an accelerated systemic aging feature by overexpressing all filoproteins, wherein an overexpression mode comprises an ERT2Cre-loxp gene expression mode in an adult biological system; wherein the accelerated systemic aging feature induced by the filoproteins includes an adult biological system that ages naturally similarly to a nighttime biological clock; and the adult biological system in which the ERT2Cre-loxp gene expression mode is applied is a mouse.

2. The application as described in claim 1, wherein the cofilin is a non-muscle cofilin (type 1 cofilin, Cofilin-1).

3. The application as described in claim 1, wherein the ERT2Cre-loxp gene expression pattern comprises a Cre-ERT2 genotype having an inducible promoter Cre-ERT2 and a Cre-ERT2 (+) / 3XFLAG-Cfl1 (+) module, wherein the 3XFLAG-Cfl1 (+) module contains a loxP-emGFP-loxP sequence nested in the proximal segment of the 3XFLAG-Cfl1 gene, driven by the ActB driver, and after the loxP-emGFP-loxP is popped out by treatment with tamoxifen (TAM), the 3XFLAG-Cfl1 will continue to be expressed.

4. The application as described in claim 3, wherein the cutting protein is an exogenous cutting protein and has a 3XFlag sequence marker for distinguishing an endogenous cutting protein, wherein the 3XFLAG peptide sequence is DYKDDDDK repeated three times to distinguish the expressed cutting protein as an exogenous protein.

5. The application as described in claim 3, wherein the administration of the tamoxifen can be obtained by feeding the adult biological system with a feed containing the tamoxifen for 21-35 days.

6. The application as described in claim 5, wherein after confirmation that exogenous fibrinogen has been induced to appear in the adult biological system, the accelerated systemic aging characteristics can be exhibited in 3-4 weeks.

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