Animal model for obese rats and establishing method

A construction method and gene technology, applied in the field of animal genetic engineering and genetic modification, can solve the problems of lack of stem cell lines, inability to fully simulate the real situation of human diseases, time-consuming and other problems

Inactive Publication Date: 2016-11-23
SUZHOU RUIQI BIO PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The whole process is not only time-consuming, but work on transgenic animals has been primarily carried out in mice due to the lack of stem cell lines for various

Method used

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  • Animal model for obese rats and establishing method
  • Animal model for obese rats and establishing method
  • Animal model for obese rats and establishing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 is aimed at the construction of the CRISPR-Cas9 expression system of Lep ( figure 1 )

[0031] 1. According to the sequence of rat Lep and LEPR (leptin receptor), design sgRNA and obtain the sequence information of sgRNA. The DNA sequence of the sgRNA specifically targeting the second exon of the Lep gene is shown in SEQ ID NO.1 and SEQ ID NO.2; the sgRNA specifically targeting the second and third exons of the Lepr gene, The DNA sequences thereof are respectively shown in SEQ ID NO.3 and SEQ ID NO.4. Among them, SEQ ID NO.1 and SEQ ID NO.2 target SEQ ID NO.5 (SEQ ID NO.5 is a part of the LEP gene), while SEQ ID NO.3 and SEQ ID NO.4 target SEQ ID NO. 6 and 7 (SEQ ID NO. 6 and 7 are part of LEPR gene, respectively). Each sequence of SEQ ID NO.1-4 is a double-stranded DNA formed by annealing. Take SEQ ID NO.1 as an example: Synthesize positive and negative single-stranded Seq ID No1-ss and Seq ID No1-as respectively, and then anneal to form SEQ ID NO.1. ...

Embodiment 2

[0041] Example 2 In vitro transcription

[0042] Use T7-Cas9 PCR and T7-sgRNA PCR products to perform in vitro transcription mediated by T7 promoter, that is, use T7 promoter as the promoter of in vitro transcription, and use RNA polymerase to realize the transcription process from DNA to mRNA in vitro. The specific method For: use mMESSAGE mMACHINE T7ULTRA kit (Life Technologies) to transcribe T7-Cas9 PCR products in vitro, and use MEGAshortscript T7 kit (Life Technologies) to transcribe T7-sgRNA PCR products in vitro. The mRNA produced by transcription was purified. The specific method was: Cas9 mRNA was purified by MEGAclear kit (Life Technologies), sgRNAs were purified by ethanol precipitation, mRNA was dissolved in pure water, and the concentration of purified mRNA was measured by spectrophotometer. T7-Cas9 PCR primers are shown in Table 4, T7-sgRNA PCR primers are shown in Table 5, gRNA-Lep1-F, gRNA-Lep2-F, gRNA-LepR1-F, gRNA-LepR2-F are respectively for SEQ ID NO.1- Th...

Embodiment 3

[0051] Example 3 Using the CRISPR-Cas9 system mRNA for Lep / LEPR to produce gene-targeted rats

[0052] 1. Pronuclear injection and embryo transfer

[0053] The pronuclear stage fertilized eggs of rats were taken, and the premixed Cas9mRNA / sgRNA mixture (the final concentration of Cas9mRNA was 100ng / μl, and the final concentration of sgRNA was 50ng / μl) was injected into the cytoplasm of rat fertilized eggs using a microinjector, and then Transplant into the oviduct of the recipient mother mouse to produce gene-targeted rats, and the injection volume is 0.5-1ul.

[0054] 2. Identification of gene targeting rats

[0055] After the birth of the surrogate mother mouse, cut off about 1 cm of mouse tail when the offspring grow to 2 weeks old, digest with proteinase K at 55°C, and extract the mouse tail genome by phenolform extraction. Using the mouse tail genome as a template, primers were designed for the 2nd exon of the Lep gene and the 2nd and 3rd exons of the LEPR gene for ampl...

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Abstract

The invention relates to an animal model for obese rats and an establishing method, and belongs to the field of animal genetic engineering and genetic modification. The animal model for obese rats and the establishing method are characterized in that rats with LEP and LEPR genes knocked out are obtained through the CRISPR/Cas9 technology. The rats with LEP and LEPR genes knocked out have a typical obesity characteristic, the reliable animal model can be provided for studying the obesity disease of people, and the rats with LEP and LEPR genes knocked out are expected to become a standard experiment animal of obese rats.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a rat animal model of obesity based on gene knockout technology and a construction method. Background technique [0002] With the improvement of living standards, obesity (obesity), as a chronic disease, has a sharp increase in incidence and has become a worldwide medical problem. Obesity is also closely related to diabetes, and 70% to 80% of diabetic patients over the age of 40 were already obese before the disease. At the same time, obesity is also accompanied by symptoms such as hyperlipidemia, hypertension, and abnormal glucose tolerance, and has become the main cause of arteriosclerosis. Leptin (Lep) is a hormone secreted by fat cells discovered in 1994. Through the Leptin receptor (Lepr) in the central nervous system, Lep can regulate various physiological functions and individual behaviors including blood sugar concentration,...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/89A01K67/027C12N15/16C12N15/12C12N5/10
CPCC12N15/63A01K67/0276A01K2217/075A01K2227/105A01K2267/0362C07K14/5759C07K14/72C12N5/0602C12N15/89C12N2510/00
Inventor 祝献民范国平曾维鸣
Owner SUZHOU RUIQI BIO PHARMA CO LTD
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