Construction and gene identification of myeloid specific Spi1 knockout mice / 安徽医科大学学报

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 @#According to the principle of CRISPR/Cas9 technology and C re/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 microinj ected 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 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/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 proviral 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 Spi1 flox/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 .

Breeding and genotyping of T lymphocyte-conditional Spi1 knockout mice / 安徽医科大学学报

Objective @#To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in- vestgation of the specific role of Spi1-encoded protein PU. 1 . @*Methods @#The Lck-Cre mice were mated with Spi1 flox/flox mice to obtain Lck-Cre ×Spi1 flox/flox mice (T lymphocyte-specific Spi1 knockout mice) , and the genotype was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis . Magnetic beads were used to sort out the splenic T lymphocytes , and the knockdown efficiency of PU. 1 in T cells was detected by Western blot , quantitative real-time PCR ( qPCR) and flow cytometry. @*Results @#The Lck-Cre ×Spi1 flox/flox mouse genotype was stably inherited . Compared with Spi1 flox/flox mice , the expression level of PU. 1 was significantly reduced in splenic T cells of Lck-Cre ×Spi1 flox/flox mice . @*Conclusion @#In this study , the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology , which provided a reliable an- imal model for the subsequent experiments of the specific role of PU. 1 in T cell-related diseases .

Construction and identification of recombinant adenovirus vectors carrying the human transcription factor PU.1 gene / 医学研究生学报

Objective PU.1 plays a key role in innate immune function in the alveolar macrophage.This study was to con-struct and identify recombinant adenovirus vectors carrying the human transcription factor PU.1 gene. Methods The recombinant shut-tle plasmid was obtained from the PU.1 gene ( SPI1) and eukaryotic expression vector that digested by restriction enzymes and connected by T4 DNA ligase.The target fragment SPI1-IRES-EGFP was amplified by PCR.The product was cloned into the intermediate pDONR221 and then recombined with the adenovirus backbone plasmid pAd/CMV/V5-DEST to form a recombinant adenovirus vector. The recombinant adenovirus vector was linearized by PacI and then transfected into human embryonic kidney (HEK293) cells to obtain the recombinant adenovirus pAD-SPI1-IRES-EGFP, which was then propagated in HEK293 cells, filtered and purified to obtain high-con-centration adenoviruses.The adenovirus titer was determined by TCID 50 assay.The PU.1 gene expression in the HEK293 cells was con-firmed by fluorescence microscopy and real-time qPCR. Results PCR amplification, restriction digestion and sequencing analysis showed the recombinant adenovirus carried the correct PU.1 gene.The final virus titer, calculated by TCID 50, was 8 ×1011 IU/mL. Green fluorescence was observed under the fluorescence microscope. Real-time qPCR confirmed that the expression of PU.1 mRNA was increased by 2189.93 folds. Conclusion The recombinant adenovirus vector carrying the PU.1 gene was constructed and obtained successfully, which could contribute to further studies of the influence of PU.1 overex-pression on the innate defense against Aspergillus fumigatus.

Transcriptional Analysis of the iagB within Salmonella Pathogenicity Island 1 (SPI1)

HilA is a central regulator of Salmonella pathogenicity island 1 (SPI1), which is necessary for host invasion by Salmonella and induction of gastroenteritis. The iagB lies downstream of hilA and is thought to be co-transcribed with hilA, but iagB expression has not yet been analyzed directly. In this study, iagB expression in various mutant strains was measured to determine whether the expression pattern was similar to that of hilA. A β-galactosidase assay revealed that iagB expression was greater under shaking than standing culture condition. iagB expression was decreased in relA/spoT and ihfB mutants but not in luxS mutant, in line with previous reports on hilA expression. The hilA and iagB mRNA levels decreased by approximately 2-fold in arcA mutant grown aerobically and increased by approximately 10-fold in fnr mutant grown anaerobically. Although the fold changes in hilA and iagB mRNA level differed in hfq mutant strain, the patterns of time- and Hfq-dependent regulation were similar for both genes. Thus, iagB and hilA exhibited similar expression patterns in various mutational backgrounds and under different growth condition.