Targeted Knockout Of βB2-crystallin Gene In The C57BL/C Mice Induces Cataract And Subfertility

Crystallins,membrane protein and cytosteletal protein are the main compositon proteins of the len.Crystallins is water-soluble,but cytosteletal protein and membrane protein and polymerized crystallins are urea-soluble.90%of the water-soluble proteins in the heman eye lens are the crystallins:α,βandγcrystallin.The study of the function ofα-crystallin is widespread and the molecular chaperone of it was generally accepted.But the study toβ-crystallin is ralatively less. The concentration ofβB2-crystallin is not only higher than otherβ-crystallin but also increase with age.So the function of it is deserved to research.Crystallin gene knockout is an important method to study the function of crystallin.Some research have find that theα-crystallin gene knockout can lead to cataract,so do theαorβ-crystallin gene mutation.It has not been reported the animal model ofβB2-crystallin gene knock out all over the world.So we chose the crystallin as the target and set up the animal model so as to research its function on maintaining the transparency of lens and its extralenticular functions.In our study,pattern electroretinogram was used to study the function of retinal ganglion cell after the gene knock out.The lens ofβB2-crystallin gene knockout mice was studied in this article.We examined the in vitro construction of len and analysis the function ofβB2-crystallin in keeping transparency of the lens and observed influence on reproduction of the mouse with targeted knockout of the mouseβB2-crystallin gene compared with the wild tipe.Methods:1.Generation and verification ofβB2-crystallin gene knockout mice.βB2-crystallin gene knockout mice were produced by the in Genious Targeting Laboratory.The model of mice were 10 heterozygote which have been breed in the central lab and have reproduced over 30 generations.Gene expression were characterized by PCR and western blot. 2.The structure and character of lens inβB2-crystallin gene knockout miceFresh lens from 8-week-old gene knockout and wild type mice were removed from the eyeballs and cultured in TC-199 medium for 2 hours in a CO₂ incubator,and then the gene knockout mice were screened for the degree of opacity with dark field biomicroscopy.Microstructure of lens was analyzed by hematoxylin and eosin stain and immunohistochemistry.By studying cortex of different ageβB2-crystallin gene knockout mice lens,we observed the influence to development of cataract.3.Fertility were studied using knock out ofβB2-crystallin mice and the wild typePattern electroretinogram was used to study the function of retinal ganglion cell after the gene knock out.We used Western Blot,PCR and immunohistochemistry to detect mRNA ofβB2-crystallin gene,βB2-crystallin outside the len(retina,brain, spinal cord,ovary and testis) although it was expressed at lower levels in several extralenticular locations.Through the babies of the mouse cipher,Sperm count,testis weigh,using morphshape and hematoxylin and eosin stain observation method,we studied the reproduction function of the mice of targeted knockout of the mouseβB2-crystallin gene compared with the wild tipe.RESULTS:1.Western blot failed to detect anyβB2-crystallin in the lens ofβB2-crystallin gene knockout mice.2.The gene knockout mice were screened more severe opacity with dark field biomicroscopy.Hematoxylin and eosin stain and immunohistochemistry found that theβB2-crystallin was located in subcapsular of the cortex and the cortex was thiner in the gene knockout mice with ageing.3.The vibration of the Pattern electroretinogram was lower after gene knock out. Through the mice the baby cipher,Sperm count,testis weigh,we found that the productivity were lower in the gene knock mice.Using morphshape and hematoxylin and eosin stain,we observed smller shape and structure chaos in the mice with targeted knockout of theβB2-crystallin gene compared with the wild tipe.Conclusions:1.Targeted knockout of the mouseβB2-crystallin gene can induce cataract which is located in the subcapsular,and then the cortex of the lens grow thinner with age.2.Targeted knockout of the mouseβB2-crystallin gene induces subfertility...