| Pigs and rabbits are important agricultural economic animals and biomedical animals.It is very important to obtain gene modified pigs and rabbits models by editing genes of pigs and rabbits efficiently and accurately.In the aspect of agricultural breeding,gene modified pigs and rabbits can successfully improve the growth speed,production,disease resistance and stress resistance of animals,and greatly accelerate the cultivation process of new excellent varieties.In the field of biomedicine,due to the interspecific differences,only using mice,rats and other small rodent models can hardly completely simulate various complex human life processes,such as the simulation of human early embryonic development,the growth and development of specific tissues and organs,and the occurrence and development of human diseases.Pigs and rabbits are ideal large animal models.Comparing with primate model,Pigs and rabbits have shorter reproductive cycle,more litter,less ethical and moral restrictions.Compared with rodents,pigs and rabbits are closer to human in anatomy,physiological and biochemical characteristics,nutritional metabolism and immune system.Many studies cannot be carried out directly in the human body,and genetically modified pig and rabbit models can be generated for more extensive experimentation and evaluation.In addition,the modified pigs and rabbits models have an important value in the function research of specific genes in the role of tissue or organ development and other dynamic processes.Cell paper from Professor He Xi of Harvard University confirmed that Tiki1 does play a decisive role in the anterior part development of Xenopus.While the function and mechanism of Tiki1 gene in mammalian system has not been reported.Because Tiki1 gene is absent in rodents such as mice,it is impossible to use rodent models to study the role of Tiki1 gene in mammalian development.In our laboratory,whole embryo in situ hybridization was carried out on the pig embryos of 12-15 days at the primitive streak stage to the neural groove stage and the results showed that Tiki1 gene was also expressed at the front edge of the embryo.Whether Tiki1 plays a role so vital in mammals as in xenopus in head induction during embryonic development needs to be verified.Gene knockout is one of the main approaches to study gene function.The traditional gene knockout technology is homologous recombination,but it is extremely difficult to obtain the desired gene target animal by homologousrecombination combined with somatic cell nuclear transfer technology(SCNT)for pig which has not successfully established embryonic stem cells or induced pluripotent stem cells that can be inherited by germline and has low cloning efficiency.Tiki1 gene targeting pig model was failed to be obtained in our laboratory using the homologous recombination technology due to its low efficiency.In recent years,nuclease gene editing technologies including ZFN,TALEN and CRISPR/Cas9 opened up a new channel for gene modification of porcine and rabbit species which has not successfully established embryonic stem cells or induced pluripotent stem cells that can be inherited by germline and has low cloning efficiency,and greatly accelerated the progress of generation and related studies of gene editing pig and rabbit models.However,the complex procedure,low efficiency and high cost of ZFN restricts its application in gene modified pigs and rabbits.In order to explore the functional differences of Tiki gene among different species,this study constructed Tiki knockout pigs and rabbits by using TALEN and CRISPR / CAS9 techniques.In this study,two pairs of TALENs plasmid were designed for the exon 4 of pigs' Tiki1 gene,and the activity of TALENs plasmid was measured in vitro at the level of porcine parthenogenetic embryos.The target efficiency and the effect on embryo development of TALENS plasmid injection was compared.The results showed that:with the increase in TALENs' injection concentration,the target efficiency also increased and the effect of TALENs' injection concentration on embryonic development in vitro was not obvious.86 cell clones were obtained by PFFs transfection with talens plasmid,6 of them were identified as positive cell clones by sequencing,and then four Tiki1 single allele targeting pig models were successfully constructed by somatic cell nuclear transfer(SCNT).In this study,X-ray Computed Tomography(CT),Magnetic Resonance Imaging(MRI),pathological analysis and physiological and biochemical analysis was used to analyze the phenotype of the Tiki1 mono-allelic knockout pig model.The analysis revealed that no Tiki1mono-allelic knockout pig model was found to have any phenotype similar to that on Xenopus.Taking into account the interspecific difference and the short experimental period of low-cost rabbits,this study successfully generated the Tiki1 mono-allelic knockout rabbit model by using TALEN technology.The results showed that no Tiki1mono-allelic knockout rabbit model was found to show any phenotype similar to that on Xenopus.As the target efficiency of TALEN is not high enough,in this study,the pigs and rabbits models constructed by TALEN technique are all Tiki1 mono-allelic knockout.In order to obtain the Tiki1 bi-allelic knockout pig model in one step,this study used CRISPR/Cas9 technology to design and construct the target plasmids to transfect pigfetal fibroblasts.A total of 8 positive single cell clones were identified by cell screening,PCR amplification and sequencing.Subsequently,720 recombinant embryos were constructed using somatic cell nuclear transfer technology and implanted into 3 surrogate sows respectively in this study and 12 Tiki1 bi-allelic knockout pig models were successfully obtained.In the follow-up study,the behavioral phenotype analysis of the Tiki1 bi-allelic knockout pig model was performed using animal gait footprint experiments and treadmill acceleration and constant-speed running behavior experiments.The results showed that no Tiki1bi-allelic knockout pig model was found to be similar to that on Xenopus Phenotype.Considering that Tiki genes include Tiki1 and Tiki2,there may be a synergistic or compensatory mechanism between the two.Because the rabbit experiment has a lower cost and a shorter cycle than the pig experiment,this study prefers rabbits as the research object.Using the CRISR/Cas9 technology,the Tiki1 and Tiki2 simultaneous biallelic knockout rabbit models were directly obtained in one step.As a result,the Tiki1 and Tiki2 simultaneous biallelic knockout rabbit models were not found to have a phenotype similar to that on Xenopus.In summary,none of the Tiki gene knockout pig and rabbit models of various genotypes obtained in this study were found to have similar phenotypes to those of xenopus,and both pigs and rabbits could survive and reproduce continuously.This study confirmed that the Tiki gene has differences in functions between Xenopus and mammals such as pigs and rabbits.The Tiki gene is indeed not a crucial gene for the development of the head of early embryos and even the growth,development and reproduction of individuals in mammals such as pigs and rabbits.It successfully clarified that the relationship between the Tiki gene and the head development of early embryos in mammals such as pigs and rabbits is different from Xenopus. |
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