Construction and validation of Hrd1 transgenic mice

Authors: Yang Shuo,  Liu Lei,  Wan Xing,  Wang Wenfeng,  Wang Juan,  He Heng,  Lei Lei,  Li Bin
DOI: 10.3760/cma.j.issn.2095-0160.2015.02.004
Published 2015-02-10
Cite as Chin J Exp Ophthalmol, 2015,33(2): 115-117.

Abstract                              [Download PDF] [Read Full Text]

Background 

Endoplasmic reticulum stress plays an important role in diabetic retinopathy (DR).Hydroxymethyl glutaric acyl coenzyme A reductase degradation protein 1 (Hrd1) is a key protein in endoplasmic reticulum-associated degradation (ERAD),and can reduce the excess endoplasmic reticulum stress.

Objective

This study was to generate Hrd1 transgenic mice and offer the animal model with Hrd1-high-expression for further pathogenesis research of DR.

Methods

The construct was generated by inserting the murine Hrd1 cDNA into a vector of systemic expression.The Hrd1 recombinant plasmids were then linearized,purified and microinjected 3 pl into 685 fertilized C57BL/6 mouse eggs,and the eggs were then transplanted into the pseudopregnant C57BL/6 mice to produce Hrd1 transgenic mice.Born transgenic founders were genotyped by PCR.The positive mice were mated with wild-type mice and reproduced generation was confirmed by PCR.

Results

The recombinant Hrd1 vector was constructed successfully,and the pRP.ExBi-EF1a-Syvn1-IRES-eGFP plasmid sequencing results showed that the cDNA sequence was correct.There were 598 survival-fertilized eggs that accepted Hrd1-pcDNA microinjection.Then the ova were implanted into the pseudopregnant C57BL/6 mice and got 41 mice pups.After PCR detection,eight F0 generations Hrd1 mice were obtained,and the physiological states of the F0 mice were normal.PCR result showed positive bands of 339 bp,and the gene type can be passed on to offspring F1.

Conclusions

This study demonstrates the successful generation of Hrd1 transgenic mice.

Key words:

Hydroxymethyl glutaric acyl coenzyme A reductase degradation protein 1; Mouse/Transgenic; Endoplasmic reticulum stress; Diabetic retinopathy

Contributor Information

Yang Shuo
Refractive Treatment Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Liu Lei
Wan Xing
Wang Wenfeng
Wang Juan
He Heng
Lei Lei
Li Bin
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Updated: June 8, 2023 — 8:48 am