A Skin-specific Knockout Mouse Model Of Ppp2ca Was Established Using Zinc Finger Nucleases And Related Research

PP2A phosphatases family is the largest serine/threonine phosphatase in mammalian.The catalytic subunit C of PP2A plays an important role in the maintenance of PP2A catalytic function in higher eukaryotes.A subunit and C subunit are respectively consisted of a and ? subtypes.All subunit combinations to create a variety of PP2A holoenzyme and they have different substrate specificity different subcellular localization as well as different tissue-specific.PP2A regulates many biological processes including DNA replication,transformation of cancer,tumor suppressor,nutritional sense,cell cycle progression,RNA transcription,apoptosis and RNA shear.Zinc Finger Nucleases(ZFNs)as a gene editing technology is widely used in basic medicial research.By designing specificity upstream and downstream ZFPs bind to the target position in genomic DNA.FOKI form a dimer and cut target sites induce double strand break(DSB)which can be repaired by error-prone non-homologous end joining(NHEJ)creating mutations can lead to the gene losing activity.In this research,we got a kind of consecutive three bases(GTG)deletion at the end of the third exon of Ppp2ca.The three bases just a codon of Valine(Val)159.In FOUNDER group,we never found a homozygous mutation mouse.Compared with other mutant type,one codon absence can not change the order of amino acids in the posterior segment.It shown that systemic knockout Ppp2ca can cause embryos death.We crossed Val159 muant genetype mice and no homozygosis mice was identified(n?105).So we suppose 3 bases deletion and inactive Ppp2ca and homozygous lead embryonic lethality.According to this,we conclude that lack of Val159 of Ppp2ca can cause Ppp2ca dysfunction and achieve the purpose of Ppp2ca knockout.Tertiary structure prediction shows Val159 mutant Ppp2ca structure is significantly changed.PP2A plays an important role in development.It reported that systemic knockout PP2AC can cause homozygous mouse embryonic lethal at E6.5.Conditional knockout PP2AC in heart tissue can cause heart failure and conditional knockout PP2AC in male mice testicular tissue can cause infertility.But so far,PP2A function in the skin is rarely studied.In this research,we used ZFNs to build absence of Val 159 of Ppp2ca mutant mice model.The lack of Val159 can cause Ppp2ca spatial structure change,and these changes may lead to its biological dysfunction,or even loss of original features.To this end,we generated a Ppp2ca conditional knockout(CKO)mouse with a loxP system to study the function of Ppp2ca in the epidermis system.We use Krt14-Cre transgenic mice to obtain Ppp2ca knockout in the epidermis.Krt14-Cre mice express Cre recombinase under the control of the human keratin 14(Krt14)promoter.It is an effective tool for generating tissue-specific targeted mutants to study the function of developmentally critical genes in the ectoderm and its derivatives.The findings of this research can be used in future studies of genetic dermatosis.In this reasearch,we can get the following 3 conclusion:?Ppp2ca 159 Valine plays an important role in the maintenance of PP2A function,it is due to Val159 absence inducing Ppp2ca protein tertiary structure changed so as to affect the functionality of Ppp2ca.?PP2A play an indispensable role in maintaining normal skin development.Knockout Ppp2ca in mice skin induce abnormal development of the skin in which cell apoptosis was not found.