Using Recombineering And CRISPR/Cas9 To Construct SCN9A Humanized Mice

The Nav1.7 is a new target for screening new analgesics with less side effects.Development of an effective drug specific to this sodium ion channel has become a research hotspot of pharmaceutical companies.Mouse genes and human genes are highly homologous.Therefore mice have been one of ideal animal models for the pre-clinical research of drugs for human diseases.However,due to the differences between proteins of human and mouse,many projects face a high risk of failure after entering the clinical research.Human SCN9A gene encodes the functional subunit of Nav1.7.The efficient development of analgesics targeting Nav1.7 requires the construction of SCN9A humanized mice.Human SCN9A gene is about 180 kb.There is not any BAC in the human genome BAC resouces covering this entire gene.The BAC sticthing to include the intact gene in one vector became the prerequisite for the construction of SCN9A humanized mice.Using recombineering,SCN9A gene was successfully stitched to a single BAC that is around 308 kb.At the same time,the inverted repeats identified by Sleeping Beauty(SB)transposase were added to both sides of the BAC vector.The Venus fluorescent protein was linked to the carbon terminal of human SCN9A protein by the 2A like peptide which has a self cleavage function.Because of the above design,we achieved large size BAC transgene by transposition without BAC vector.The Venus fluorescent protein reporter allow us to map the expression profile of human SCN9A protein in mice.The modified BAC and SB transposase expression plasmid were introduced into mESCs by liposome transfection.The 300 kb DNA segment containing human SCN9A gene was successfully integrated into the mESCs genome.The homologous arms for making the mouse Scn9a gene targeting construct was directly cloned from the genomic DNA prepared from mESCs.We achieved quick construction of the targeting vector with long homologous arms.The gene targeting using long homologous arms was assisted by CRISPR/Cas9.The DNA cassette for mCherry fluorescent protein expression was inserted at the downstream of the mouse Scn9a gene promoter,replacing the critical exons and inactivating the function of the targeted gene.Cherry fluorescent protein can be used to map the expression profile of the mouse SCN9A protein.PCR and ExoCET were combined for genotyping of the targeted cells.This made the results more accurate and reliable.We obtained both monoallelic and biallelic Scn9a gene knockout mESCs.The monoallelic knockout mESCs will be used to construct the humanized mice.The biallelic Scn9a gene knockout mESCs can be induced to neuralstemcells(NSCs)for functional research of SCN9A at the cell level.SCN9A mutations are closely related to various neuropathic pain diseases,such as Congenital Indifference to Pain(CIP),Inherited Erythromelalgia(IEM),Paroxysmal Extreme Pain Disorder(PEPD),and Small Fiber Neuropathy(SFN).Among them,G856D and A1632E mutants are complicated in clinical phenotypes and electrophysiological characteristics,showing the combined symptoms of IEM and SFN,IEM and PEPD.Therefore,the construction of SCN9A humanized mice containing G856D or A1632E mutation are more important.In this study,using recombineering together with the cdB counter-selection marker,we successfully generated G856D and A1632E mutations which are related to the pain hypersensitive.The study show the first example of using stitched large BAC for transposition of mESCs.The human SCN9A gene was integrated into the mouse genome.This created favorable conditions for functional research of human SCN9A protein and the molecular mechanism of pain.The elaborated design of the mouse Scn9a gene targeting and the human SCN9A gene BAC transgenesis using different fluorescent reporters also allow us to map the expression profiles of human and mouse SCN9A protein in mice,which provide important basis of application of this mouse model for analgesics screening.Using the SCN9A humanized mice or the SCN9A humanized mice containing G856D or A1632E hypersensitive mutation for analgesics screening will largely reduce the data bias caused by species differences,thus increase the success rate after entering into the clinical trials.