ObjectiveTo humanize the variable region of the IL9-2G1 rabbit monoclonal antibody （IL9 2G1 mAb） and to evaluate its biological activity. MethodsThe humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region （VH） and light chain variable region （VL） of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell （CHO） expression system. Enzyme-linked immunosorbent assay （ELISA） was used to qualitatively analyze the antigen-binding activity of the antibodies. After screening the optimal humanized antibody candidate， surface plasmon resonance （SPR） was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 （JAK1/STAT3）， while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 （IL-9） secretion. ResultsPurification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant （KD） of M501042-OHFL against the analyte M30903 was 1.33×10^-7 M， which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 （p-JAK1） and phosphorylated STAT3 （p-STAT3） were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group （P<0.01）. The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group （P<0.01）. ConclusionHumanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody， and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed， which provides an experimental basis for subsequent research.

ObjectiveTo construct an interleukin-9 （Il9） gene knockout mouse model on a C57BL/6N background， establish a reliable genotyping method， and provide a standardized animal model for investigating Il9-related immune mechanisms and diseases. MethodsIl9 knockout mouse strain （C57BL/6N-Il9^em1Cya） was generated using conventional gene knockout technology. The method for DNA extraction from mouse tails was optimized to ensure template quality. Two sets of specific primers （F1/R1 and F1/R2） were designed to amplify target fragments by PCR， and mouse genotypes were determined by agarose gel electrophoresis. Specifically， primer pair F1/R1 targeted the knockout sequence with an expected product of 465 bp， while F1/R2 targeted the wild-type sequence with an expected product of 703 bp. Subsequently， Western blot was performed to verify the knockout efficiency of Il9 in primary immune cells and key organs of the offspring mice， and flow cytometry was used to assess the effects of Il9 deficiency on the function of the immune system in major immune cell populations. ResultsIl9 knockout mice were successfully bred and genotyped. PCR identification showed that homozygous mice （-/-） exhibited only a 465 bp band， wild-type mice （+/+） displayed only a 703 bp band， and heterozygous mice （+/-） showed both bands simultaneously. Meanwhile， Western blot analysis revealed significantly decreased IL-9 expression in various organs of the offspring mice （P<0.001）. Flow cytometric results demonstrated that Il9 deficiency exerted no obvious effects on the proportions of peripheral blood lymphocytes， splenic lymphocytes， and peritoneal macrophages. ConclusionAn Il9 gene knockout mouse model on a C57BL/6N background is successfully constructed. The established PCR genotyping system is highly efficient and accurate， which can effectively distinguish mice of different genotypes. This model provides a stable and reliable experimental basis for subsequent functional studies of Il9 and exploration of the mechanisms underlying Il9-related diseases.

ObjectiveTo construct a chimeric antigen receptor macrophage （CAR-M） targeting interleukin-13 receptor α （IL-13Rα） and intracellular domain integration， which can double activate the signal adaptor protein OX40L of macrophages and T cells. MethodsThe CAR molecule targeting IL-13Rα （IL-13Rα-OX40L-CAR） with OX40L as the intracellular signal domain was designed and synthesized， and it was cloned into the adenovirus expression vector. After THP-1 cells were induced to differentiate into macrophages （THP-1-M） by phorbol ester， the cells were infected with recombinant adenovirus to construct THP-1 macrophages stably expressing green fluorescent protein （GFP） labeled anti-IL-13RαCAR. The expression efficiency of CAR molecules was monitored by flow cytometry. The empty vector group was used as the negative control， the CD3ζ group was used as the positive control， and the CD3ζ-OX40L group was used as the experimental group. The phagocytic function of macrophages in each group on IL-13Rα high expression glioma cells （U251） and low expression glioma cells （T98G） was detected by in vitro co-culture experiments. ResultsCAR adenovirus was efficiently transfected into THP-1-M to obtain anti-IL-13RαCAR-M. After co-cultured with glioma cells with different expression levels of IL-13Rα， the results of flow cytometry showed that there was no significant difference among the three groups for T98G cells with low expression of IL-13Rα. After co-culture with U251 cells with high expression of IL-13Rα， the CD3ζ group showed enhanced phagocytosis compared with the empty vector group （P<0.05）. Compared with the CD3ζ group， the phagocytosis ability of the CD3ζ-OX40L group was further enhanced （P<0.05）. ConclusionAnti-IL13Rα- OX40L-CAR-M is successfully constructed by constructing IL-13Rα-OX40L-CAR adenovirus and transfecting PMA-induced differentiated THP-1-M. It can engulf glioma cells with high expression of IL-13Rα by specific targeting， and shows activation of intracellular domain superior to traditional CD3ζ.

ObjectiveTo construct B cell conditional knockout Spi1 gene mice and analysis their genotypes to provide an animal model basis for disease pathogenesis and drug target research. MethodsMb1-cre transgenic mice were crossed with Spi1^flox/flox mice. PCR combined with agarose gel electrophoresis was performed for genotyping， and Mb1-cre×Spi1^flox/flox homozygous knockout mice were screened out. Flow cytometry was used to detect the expression of PU.1 protein in various immune cell subsets to verify the specificity of gene knockout. ResultsPCR identification results showed that when using flox primers for detection， only a 220 bp band was amplified， indicating a genotype of Spi1^flox/flox in the mice. When using Mb1-Cre primers for detection， a 383 bp band was amplified， indicating a genotype of Mb1-cre×Spi1^flox/flox in the mice； Flow cytometry results showed that compared to Spi1^flox/flox mice， Mb1-cre×Spi1^flox/flox mice exhibited significantly reduced PU.1 expression levels in bone marrow-derived mononuclear cells， peripheral blood mononuclear cells， and spleen-derived B cells. Moreover， PU.1 exhibited normal expression in other immune cells， such as CD4⁺ T cells and macrophages. ConclusionBy utilizing the Cre/LoxP system and CRISPR/Cas9 technology， this study successfully generates mice with B cell-specific conditional knockout of the Spi1 gene， providing a reliable animal model for in-depth exploration of the specific role of PU.1 in B cell-related diseases.

Objective: To constructCSF1R+/-mice and to analyze their genotypes, so as to provide animal model basis for disease pathological mechanism and drug target. Methods : A linearized targeting vector was designed according to Cre/Loxp system. A Loxp site was inserted upstream of the 5th exon of theCSF1Rgene, and a neomycin resistance box with Loxp sites on both sides was inserted downstream of the 5th exon. The linearized targeting vector was electroporated into embryonic stem cells. The correctly targeted embryonic stem cells were injected into the blastocysts of C57BL/6J mice to obtain chimeric mice, which were bred with Zp3-Cre mice. The newborn mice were numbered 9-14 days after birth and their tails were cut. The DNA of the mice was extracted, and the genotype of the mice was identified by polymerase chain reaction and agarose gel electrophoresis. The expression of CSF1R in mouse macrophages was detected by flow cytometry. The expression of CSF1R in mouse tissues was detected by Western blot. Results: The results of agarose gel electrophoresis showed that 453 bp bands were amplified in wild type mice, and 453 bp and 650 bp bands were amplified in heterozygous mice. The results of flow cytometry showed that the expression of CSF1R in peritoneal macrophages and bone marrow-derived macrophages of CSF1R heterozygous mice was lower than that of WT group(P<0.05). The results of Western blot showed that the expression of CSF1R in spleen, kidney and brain tissue of CSF1R heterozygous group was lower than that of WT group(P<0.05). Conclusion CSF1R+/-mice are successfully constructed, reproduced and identified, which provides an animal model basis for further revealing the potential mechanism of CSF1R in immune regulation.

Objective : To construct triggering receptor expressed on myeloid cells 2(TREM2) gene knockout(TREM2-/-) mice using CRISPR/Cas9 technology, to breed TREM2-/- mice and to analyze the genotype of TREM2-/- mice. Methods : CRISPR/Cas9 technology was used to selectively knock out exon 2-3 regions of TREM2 gene to construct a TREM2-/- mouse model, and the genetic background of all mice was C57BL/6J. Polymerase chain reaction(PCR) was used to identify the genotype of mice. Quantitative real-time PCR(qPCR) and Western blot were used to detect the expression level of TREM2 in major tissues of mice, and the authenticity and scientific nature of PCR identification results were verified from mRNA level and protein level. According to the sgRNA sequence, the possible off-target sites were predicted on the CCTop website, and the tail DNA of mice was extracted and amplified by PCR and then Sanger sequencing was performed to detect whether there was off-target effect in TREM2-/- mice. Results : TREM2-/- mice were successfully constructed by CRISPR/Cas9 technology, and the mice were genotyped. The results of agarose gel electrophoresis showed that the mouse genotype with only 415 bp band amplified was wild type(WT), the mouse genotype of the 449 bp band amplified only was TREM2-/-, and the mouse genotypes amplified with 415 bp and 449 bp double bands were heterozygous. qPCR results showed that compared with WT mice, the mRNA expression of TREM2 in heart and brain tissues of TREM2-/- mice was down-regulated(P-/- mice was reduced(P-/- mice. Conclusion TREM2-/- mice are successfully constructed and bred, a reliable genotype identification method is established, the genetic stability of the mouse model is verified, which will provide an important genetic animal model for the study of TREM2 gene function.

Objective : To construct a humanized mouse model of the interleukin-9 receptor(IL-9R) coding DNA sequence(CDS) gene and to verify the genotype and IL-9R expression in mice. Methods : The CRISPR/Cas9 genome editing technology was used to replace the exon 2-7 fragment of the il-9r gene in mouse embryonic stem cells with the corresponding human IL-9R sequence. After verifying the completion of the gene fragment replacement, tetraploid embryos were constructed and microinjected back into the oviducts of surrogate mice. Through surrogacy by female mice, homozygous humanized mice were obtained. DNA was extracted from the homozygous humanized mice IL-9R CDS gene, and their genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of IL-9R in the spleen and thymus of homozygous humanized mice with either wild-type(WT) or IL-9R gene humanization. Results : Gel electrophoresis after PCR amplification showed that mice with only a 1 805 bp band amplified using WT primers were wild-type, while mice with 2 553 bp and 2 340 bp bands amplified using 5KI and 3KI primers, respectively, were homozygous humanized mice with IL-9R CDS gene. Western blot results indicated that the tissues of homozygous humanized mice model with IL-9R CDS gene expressed IL-9R significantly. Conclusion The humanized mouse model with IL-9R CDS gene has been successfully constructed and characterized.

Objective: To construct microRNA(miR)-22 gene knockout(miR-22-/-) mice using CRISPR/Cas 9 technology, to breed miR-22-/- mice and to identify their genotypes. Methods : In this experiment, CRISPR/Cas 9 technology was used to construct miR-22-/- genetically engineered mice. After gene identification, the F0 generation miR-22-/- mice were mated with wild-type mice in the same litter to obtain F1 generation miR-22-/- mice. The miR-22 knockout efficiency was analyzed at the RNA level by real-time fluorescence quantitative polymerase chain reaction(qPCR). Western blot was used to detect the interaction between miR-22 and target genes. Results : miR-22-/- mice were successfully constructed using CRISPR/Cas 9 technology, gene identification was performed on the bred mice, and three stable genotypes of miR-22+/+,miR-22+/-, and miR-22-/- were identified. The real-time fluorescence quantitative PCR detection results confirmed that miR-22-/- mice showed almost no expression of miR-22 in the heart, liver, lung, kidney, spleen, and thymus tissues compared to wild-type mice in the same litter. Western blot analysis showed that the relative expression level of NLRP3 protein in miR-22-/- mouse tissues was lower than that in wild-type mice. Conclusion A miR-22-/- mouse model is successfully constructed, and stable genetic homozygous miR-22-/- mice is obtained. This indicates that miR-22 has an inhibitory effect on the downstream target gene NLRP3.

Objective: To explore the breeding and genotyping of CCR2 knockout mice, and to verify the applicability of the polymerase chain reaction(PCR) method for genotype detection of CCR2 knockout mice. Methods: The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation, the obtained F1 generation heterozygous mice were continued to be mated. DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks, the target gene fragment was amplified by PCR, and the genotypic results were determined by agarose gel electrophoresis. The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses, the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs, and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells. Results: CCR2 knockout mice were successfully bred and characterized, and three genotypes of F2 generation mice were obtained: CCR2+/+, CCR2+/-, and CCR2-/-. The offspring genotypes were identified by PCR, and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice. Flow analysis showed that CCR2 knockdown reduced the expression of CD4+T and Th1 cells in mouse spleen-derived T cells, but did not affect macrophage function. Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice, and PCR method for identifying mouse genotypes is simple, fast and reliable.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.