| Animal models are essential in biomedical research such as the pathogenesis and treatment of human diseases.Due to the differences in physiological characteristics,genetic background and immune system between humans and animals,the animal immune system will produce immune response to transplanted human cells or tissues,which greatly limits the application of experimental animals and the study of human-related diseases.therefore,the construction of immunodeficient animals and their humanized research will broaden the scope of application of animal models and promote the study of related diseases.In 2007,Takenaka reported for the first time that the polymorphism of SIRPαgene in NOD mice is conducive to the implantation of human hematopoietic stem cells,and its expression in mouse macrophages can enhance the host’s tolerance to foreign grafts.With the deepening of research,more and more evidence shows that the binding of SIRPαprotein to CD47 protein produces a"don’t eat me"inhibitory signal,thus avoiding phagocytosis of foreign grafts by phagocytes.The affinity between SIRPαprotein and human CD47 protein determines the effect of transplantation.At present,the BAC plasmid containing human SIRPαgene is usually used to humanize immunodeficiency animal models.The expression of hSIRPαprotein is beneficial to the transplantation of human cells or tissues in animals,and promotes the development of humanized animal models.However,there are no reports about hSIRPαtransgenic rabbits.Bacterial artificial chromosome（BAC）can carry 150-300kb genomic fragments,and the inserted sequence usually includes gene enhancers and other regulatory elements to maintain stable expression of the transferred gene.In this study,the BAC plasmid containing hSIRPαgene was injected into the pronucleus of rabbit fertilized eggs by microinjection,and then the injected fertilized eggs were transferred into rabbit fallopian tubes by embryo transfer.The gene humanized rabbit model was constructed,which is called NOD-like rabbit,and its indexes were detected.The results are as follows:1)Through the amino acid sequence alignment of the IgV domain of SIRPαprotein and the IgSF domain of CD47 protein in various species,it was found that the amino acid sequence homology of SIRPαIgV domain between rabbit and NOD mouse was 62%,rabbit and human SIRPαIgV domain was 68%.The amino acid sequence homology of rabbit and NOD mouse CD47 IgSF domain was 60%,rabbit and human CD47 IgSF domain was 63%.These homologies are low,so it can be inferred that the binding affinity of rb SIRPαprotein to h CD47 protein is low.2)In this experiment,79 fertilized eggs were transplanted into three recipient rabbits,two were pregnant,and a total of 14 young rabbits were born.Receptor 1（R001）and receptor 2（R002）gave birth to 5 and 9 offspring respectively.Among them,5 rabbits born to R002 receptor died one after another.At present,there are 9 surviving young rabbits.3)Because BAC-hSIRPα（208kb）plasmid is easy to break,the genetic integrity of blastocysts injected with BAC-hSIRPαplasmid（No.1-10）obtained by in vitro culture was identified by PCR method before embryo transfer.In this experiment,a total of 6 pairs of primers were designed to identify blastocysts,of which 5 pairs of continuous primers PCR were detected in No.3,4,7-9blastocysts,indicating that the injected gene had good integrity.4)Nine pairs of primers were designed across hSIRPαgene（46kb）to detect the gene integrity of ear tissues of newborn rabbits.The results showed that the degree of integration of hSIRPαgene was different in 14 newborn rabbits.Among them,9 pairs of primers in R002-6 and R002-9 rabbits were positive,while only 3 pairs of primers in R002-7 rabbits were positive.At the same time,the copy number of foreign gene was detected by absolute quantitative PCR.Among the 14 transgenic rabbits,the highest copy number of R002-9 transgenic rabbits was 1.75±0.44,and the lowest copy number of R002-7 transgenic rabbits was 0.42±0.05.It was confirmed by PCR results.5)The tissues of kidney,ileum,brain,liver,heart,stomach and spleen of transgenic rabbits were taken and the mRNA expression of hSIRPαin each tissue was detected by relative quantitative PCR.The results showed that the expression of hSIRPαgene was the highest in ileum,followed by stomach and spleen,and the lowest in liver.The mRNA expression level of hSIRPαgene in ileum,stomach and spleen of transgenic rabbits was significantly higher than that of wild type rabbits.6)In order to detect the effect of hSIRPαtransgenic on the autoimmune components of rabbits,we analyzed the components of peripheral blood cells of transgenic rabbits.The results showed that there was no significant difference in the number and proportion of immune cells such as lymphocytes and monocytes between wild-type rabbits and wild-type rabbits,indicating that the insertion of hSIRPαgene did not affect the development of immune components in rabbits.In addition,the expression of hSIRPαwas detected by flow cytometry.The results showed that hSIRPαgene was expressed in different degrees in transgenic rabbits,and the expression level of R001-2was the highest.The results showed that among the 14 newborn rabbits,hSIRPαgene was integrated in varying degrees,among which R002-9 and R002-6 transgenic rabbits had the highest degree of integration.There was no significant difference in T and B cells and other immune components between living transgenic rabbits and wild-type rabbits,and the expression level of hSIRPαprotein in R001-2newborn rabbits was the highest.With the emergence of gene editing technology,a variety of immunodeficiency(PRKDC-/-,RAG1/2-/-,IL2Rγ-/-)rabbit models have been successfully constructed.If propagated with hSIRPαtransgenic rabbit models,rabbit models similar to NOG,NRG and NSG mouse strains can be obtained,which greatly enriches the types of rabbit disease models that can be used for humanized research,so as to better simulate the human internal environment and obtain more real physiological and pathological responses. |
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