Establishment Of A Detection Method For Low Abundance FecB Gene In Sheep And Study On BMPR-1B Gene Targeting In Sheep

Sheep lambing rate is one of the main economic traits and directly related to the economic benefit of the sheep industry.The lambing rate is manipulated by multiple genes,including FecB gene witch is A746 G Mutated bone morphogenetic protein receptor lB(BMPR-1B).FecB gene is continuously been paid attention because of its improving lamb rate dramatically,Heredity steadily and accumulation mechanism in its effect.The FecB gene was mainly distributed in some high fertility breeds.Tt is verified that FecB gene is naturally present in small number of low fertility sheep breeds,it is very important and will benefit the sheep production that find and pick out the sheep carrying FecB gene and make them keep breeding.In addition,there is no report about the rapid and high-throughpu detection of FecB gene in a large group of sheep.Moreover,FecB gene is widly introduced by means of hybridization to improve the local low fertility breeds,that lead to some Adversely affect on them..The adversely affection can be avoided if the introduction of FecB gene is carried out in the way of Genome editing,not of hybridization between sheep breeds.it is a pity that there are few researches in this field at present time.[Objective]: One purpose of this study is to establish a low-abundance detection method for rapidly high-throughput screening of FecB gene in sheep,another purpose of this study is introduce FecB gene into Aletai sheep by gene targeting,in order to improve the lambing rate of the sheep,and to provide reference for other low fertility sheep.[Methods]:1 Establishment of a low-abundance detection method for FecB gene in sheepAccording to the requirements of COLD-PCR(co-amplification lower denaturation temperature-PCR COLD-PCR,)and HRM(High resolution curve analysis,HRM),FecB gene detection primers we designed and synthesized.The Genomic DNA were extracted from Hu sheep and Aletai sheep.COLD-PCR standard template products was prepared with their Amplicon DNA.The FecB genotyping and its detection sensitivity were dones with standard qPCR HRM method,320 Hu sheep were genotyped by the method,The genotype result and lambing rate were acuracily statisticed for analysising correlation between FecB gene and lambing rate.By optimizing the conditions,the COLD-PCR HRM detection method for low-abundance FecB gene was establishmented,which is Reliable and feasible.2 Preparation of FecB mutant Fetal fibroblast cell line of Aletai sheepIn this study,a pair of TALENs plasmids were designed and commissioned to the company for construction,which activity was assayed by sequenceding and HRM.The targeting vector PMD18-L-loxp-pA-noe-PGK-loxp-R-PGK-DTA-pA(named as K1)and negative targeting vector PMD18-L-loxp-pA-R-PGK-DTA-pA(named as K2)for sheep FecB gene were constructed,and An single strand oligonucleotide(SSO)was prepared,the three were used as homologous recombination DNA donors.The Aletai sheep fetal fibroblasts were isolated and detected for FECB gene and Gender.Targeting plasmid and SSO were transfected into Aletai sheep fetal fibroblast cells with a pair of constructed TALENs plasmid respectively,and pEGFP-N1 instead of DNA donor was used in carrying out parallel experiments,48 hours later after transfection,the cells were counted under the inverted microscope at high magnification,calculating the transfection efficiency according to total cells in the white light and UV light,or directly delivered to the flow cytometry fluorescence for evaluating the transfection efficiency.SSO and K2 vector were transfected into cells,monoclonal cell lines were prepared by limited dilution method.The cells transfected by K1 vector were selected by G418,the resistant monoclonals were obtained.The monoclonal cell lines were tested by PCR sequencing and gene knock-in Experiment.for FecB.3 Construction of FecB mutant embryos and individualsOvine oocytes were collected.FecB mutation positive monoclonal cells were used as donor,nuclear transfer,fusion,activation were conducted,after culturing,part of oocytes were transplanted into surrogate estrous ewes,another part were Continuously cultured for a week for observing the embryo development and counting for blastocyst rate.At the same time,the no-targeted cells were used as the donor for nuclear transfer,and the same operation was carried out as the control of embryonic development.In addition,we tried the sheep fertilized eggs microinjection method,after injecting TALENs and k2 targeting vector,the embryos were transfered,hoping to get genetically modified lamb.[Results]:1 Screening FecB gene by COLD-PCR HRM in sheepIn this study,the sheep FecB gene qPCR HRM genotyping method was established,the curve separation is clear,the detection sensitivity is 2%.the curves for 320 Hu sheep FecB genotyping are clear and accurate.Correlation analysis showed that BB genotype is more productive than B+ genotype sheep.The sensitivity of COLD-PCR HRM was 0.5%,and the repeatability was good and the results were reliable.2 Acquisition of FecB mutation positive monoclonal cellsThe TALENs plasmids which has a certain of activity in the cell,were used for downstream experiment;targeting vector sequences were verified by sequencing;on the basis of green fluorescence detection,the highest transfection efficiency of electroporation and nuclear transfection were 70.6% and 90%,Respectively.By Co-transfection of SOO and K2 targeting vectors,162 and 99 single cell clones were obtained by limited dilution method Respectively,single cell clone formation efficiency were 25.0% and 30.1%,Respectively.No FecB mutation positive cells was found by sequencing.After G418 screening in two round,5 single cell clones were obtained.Two of witch were heterozygote detected by PCR amplicon sequencing and T-A coloning sequencing.The heterozygous cells were active and used as a nuclear donor,laid a foundation for the construction of individual and embryos of Aletai sheep FecB mutation.3 The construction of Aletai sheep FecB mutant embryos and individual.By FecB mutant positive FecB cells used as donor and nuclear transplantation,a total of 89 reconstructed embryos were abtained.the cleavage rate was 69.9%,the blastocyst rate was 14.9%,and the control without targeting treatment were compared with no significant difference.After Embryo beening transferred to 14 surrogate ewes,No miscarriage or lamb were found coming into the world.In addition,44 fertilized eggs were obtained by microinjection,and the embryos were transplanted into 22 sheep.The pregnancy rate was 63.6% after 60 d.the normal birth rate was 59%.No aborted fetuses or lambs Carried FecB by sequencing[Conclusion]The COLD-PCR HRM detection method established in this study provides a convenient,low-cost,high-throughput and rapid screening technology for the sheep FecB gene.by gene targeting,we envently did not get Aletai sheep individual with FecB mutation,but abtained reconstructed embryos,witch offered materials for the study on the mechanism of Fec B and for the reconstructed sheep.