Generation Of A Miniature Pig Disease Model For Human Dwarfism(Laron Syndrome)

Laron syndrome is an autosomal recessive disorder due to the mutations of GHR gene.This mutation causes dyfunction of GH-GHR singalling pathway leading to the reduction of IGF-I in circulation,which affacts the body growth,metabolism and development.Laron syndrome is characterized as dwarfism,frontal bossing,small midface,modeate obesity and small genitalia.Biochemistrical features include high levels of GH and low levels of IGF-I in circulation,and there is no response to the administration of GH.Those clinical characters occurred in fetal and it will develop to dwarfism gradually without treatment.The only treatment for this disease is recombinant IGF-I,however,it will bring various side effects by long time administration of IGF-I to the Laron patients.So far,researchers have utilized homologous recombination to produce GHR gene knockout mouse or liver-specific GHR knockout mouse to investigate this disease,whereas the huge differences between mouse and human made it difficult to fully recapitulate human Laron syndrome.We employed gene-editing tools to knockout the GHR gene in miniature pig fibroblast due to the low efficiency of the homologous recombination in many species.First,we obtained two pairs of ZFNs for exon 6 of the GHR gene from commercial company,and the mutation efficiency of ZFNs set 2(22.5%)was higher compared with set 1(2.04%).We also designed TALENs and CRISPR/Cas9 for exon 6,and the mutation efficiencies for them were 45.2%and 26.2%,respectively.Furthermore,we designed TALENs and CRISPR/Cas9 for sequences encoding intracellular tyrosines of GHR as well as sequences in exon 10 and 3'-UTR.TALENs for those sites exhibited an efficiency ranging from 6%to 34.8%,while CRISPR/Cas introduced mutations at specific sites ranged from 8%-25.1%.To mimic the GHR mutation type of dwarf chicken in pigs,we have also co-transfected two pairs of TALENs or CRISPR/Ca9 to knock out more than 2770 bp that includes part of exon 10 and 3'-UTR sequences of the GHR.Above all,ZFNs,TALENs and CRISPR/Cas9 could introduce mutations in pig genome with a high efficiency.The-/-(GHR4bp4bp)pigs were obtained through breeding of +/-(GHR+/4bp)pigs.We obtained the GHR+2bp,GHR+/4bp,GHR+/3bp by SCNT in which no remarkable Laron phenotypes were observed.Therefore,we planned to obtain GHR double alleles knockout pigs by means of breeding.Before breeding,we cloned pig GHR transcript with a 4 bp insertion to determine if it will still maintain the function of signal transdcuction in vitro.Pig GHR transcript with a 4 bp insertion could express in DF-1 cells,while this mutated pig GHR protein located in cytoplasm instead of on the membrane by immunofluorescence assay.Additionally,the mutated pig GHR lost most signal transduction function though it could bind to bovine GH as the same level as the wild-type GHR.At last,we obtained six-/-pigs in the F2 generation.The GHR mRNA level were significantly reduced in-/-pigs compared with+/-pigs and +/+(GHR+/+)pigs.GHR protein was not detected in-/-pigs by Western blot.The body weight and body lenght of-/-pigs was significantly lower and shorter than ?/-pigs and +/+ pigs followed weaning.-/-pigs exhibited a significantly higher GH and lower IGF-I in serum,while there were no significant differences between ?/-pigs and +/+ pigs.The aforementioned results in the-/-pigs are consistent with those obtained in Laron patients.Above all,we successfully generated a Laron syndrome disease model in miniature pigs by employing ZFNs.This model will give us further data of human Laron syndrome,it will also facilitate the development of new treatments for this genetic disorder so as to bridge the gaps between mouse model and human.