Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction method and application of tshbeta gene deleted zebrafish model

A construction method and zebrafish technology, applied in the field of biomedicine, can solve the problems of drug side effects, damage to thyroid function, time-consuming and labor-intensive problems, and achieve the effect of obvious phenotype and convenient large-scale drug screening

Pending Publication Date: 2022-03-01
SUZHOU UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the current research on hypothyroidism is mostly carried out through drug treatment or thyroidectomy. The current research techniques and methods for hypothyroidism, such as performing thyroidectomy on animals, are time-consuming and laborious. The research speed is very slow and it is easy to accidentally injure experimental animals. Taking drugs to temporarily hypothyroidism will cause side effects of drugs, and it is inconvenient to damage the thyroid gland by laser and radiation to cause functional impairment

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Construction method and application of tshbeta gene deleted zebrafish model
  • Construction method and application of tshbeta gene deleted zebrafish model
  • Construction method and application of tshbeta gene deleted zebrafish model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Determine the target site according to the sequence of zebrafish tshβ in NCBI. Target sites were designed through the TALE-NT website: https: / / tele-nt.cac.cornell.edu / (Doyle et al., 2012). The present invention selects target site on the second exon of tshβ gene ( figure 1 A). The left arm sequence is GCCACCTATCATGTCTCT, and the right arm sequence is TTACTTCCAACGGCACGGA. The target site contains a SphI restriction site for subsequent identification ( figure 1 A).

[0038] (2) After the TALEN expression vector was linearized and purified by restriction endonuclease NotI, capped mRNA was synthesized in vitro by SP6 transcription kit (SP6 mMESSAGE mMACHINE kit, Ambion).

[0039] (3) Microinject 250pg capped TALEN mRNAs into each single-cell zebrafish fertilized eggs, and a pair of capped TALEN mRNAs were injected into the embryos at the same time, and then the embryos were stored in E3 solution at 28°C ( 5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2 ,0.33mM MgSO 4 ). ...

Embodiment 2

[0050] Embodiment 2 reproductive sterility model

[0051] (1) For the homozygous mutant tshβ - / - The natural spawning success rate test was carried out, and it was found that the mutant self-fertilization did not lay eggs, and the mutant male and female mated with WT respectively, but also did not lay eggs. Compared with the 91.7% spawning success rate of WT, the difference was extremely significant ( image 3 ).

[0052] (2) Use 5-month-old adult zebrafish, take 7 points a day, and use 6 fish for each sample at each time point. Homogenize the whole fish with a homogenizer, dilute with PBS solution, centrifuge at 4000g for 15 minutes at 4°C, draw the supernatant and use an ELISA kit to test for testosterone, estradiol, luteinizing hormone, follicle-stimulating hormone and gonadotropin Determination of hormone-releasing hormones.

[0053] It was found that in the male mutant tshβ - / - In , the expressions of testosterone, estradiol and follicle-stimulating hormone were signi...

Embodiment 3

[0058] Embodiment 3 skin pigment research

[0059] The pigment cells of zebrafish are mainly composed of melanocytes, yellow pigment cells and flash cells ( Figure 8 ). In our study, we found that adult tshβ - / - In , the number of melanocytes in the abdominal stripe was significantly increased, while the number of flash cells was significantly reduced near the heart ( Figure 9 ). From the top view, we also observed that in the dorsal tshβ - / - , the number and range of melanocytes were also significantly higher than those of heterozygous and wild type ( Figure 10 ).

[0060] Therefore zebrafish tshβ - / - It can also be used as a pigment model to screen the effects of various drugs on several different pigment cells, especially the melanocytes in the abdomen. Specifically, take adult zebrafish tshβ - / - , Soak the drug solution to be screened for a suitable time (such as 3dpf), take pictures under a microscope to observe the changes in melanin, and use T4 soaking as a p...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for constructing a tshbeta gene deleted zebrafish model, which comprises the following steps of: obtaining TALEN mRNA (Ribonucleic Acid) by utilizing a TALEN gene editing technology, injecting the TALEN mRNA into a zebrafish fertilized egg in a single cell period to obtain an F0 embryo, feeding the F0 embryo to sexual maturity, mating with wild type zebrafish to generate F1, screening out zebrafish capable of inheriting tshbeta mutation, and constructing the tshbeta gene deleted zebrafish model. The method comprises the following steps: selfing zebrafish which has the same mutation type and can inherit tshbeta mutation, and screening out homozygous tshbeta- / -to obtain the tshbeta gene deleted zebrafish model. The built zebrafish model has huge potential in related researches such as a thyroid disease model, a reproductive sterility model for researching the influence of thyroid on reproduction, a skin melanin model for screening the effect of drugs on melanin and the like.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a construction method and application of a tshβ gene-deficient zebrafish model. Background technique [0002] So far, various techniques to induce hypothyroidism have been widely used in experimental animals, including thyroidectomy, thyroid damage induced by absorption of radioactive iodine I, dietary restriction of iodine absorption or intake of goiter substances. For example, Bai Qingshuang et al. (Preliminary study of levothyroxine gel in the treatment of rat hypothyroidism model, Chinese Journal of Nuclear Medicine and Molecular Imaging, 2021, 41(8): 486-489. DOI: 10.3760 / cma.j.cn321828- 20201110-00407.) Established a hypothyroid model by intraperitoneal injection of 131I, and studied the therapeutic effect of levothyroxine gel on rat hypothyroidism model; Sun Chunping et al. The effect of protein expression, Chinese Journal of Clinical Health, 2010, 13(4): 382-384, ins...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/55C12N15/16A01K67/027A61K49/00
CPCC12N15/89C12N9/22C07K14/59A01K67/0276A61K49/0008A01K2217/075A01K2227/40A01K2217/15A01K2267/03
Inventor 季成
Owner SUZHOU UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com