FOXO3A基因敲除小鼠繁殖鉴定及骨髓造血干细胞表型初步分析
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摘要
目的繁育和鉴定FOXO3A基因敲除小鼠,选育的FOXO3A基因敲除杂合型小鼠用于保种,纯合型小鼠用于实验。初步分析FOXO3A基因敲除对小鼠造血细胞表型的影响,为后续FOXO3A基因对造血细胞损伤的调节研究奠定基础。方法杂合型雌鼠与野生型雄鼠交配,选育出杂合型雌鼠和杂合型雄鼠,采用一雄一雌或一雄两雌同笼合养方式进行繁育获得纯合型小鼠;繁育的幼鼠剪趾标号,剪尾提取基因组DNA,采用PCR方法进行基因型鉴定,鉴定获得100 bp/186 bp两条条带的为杂合型小鼠(FOXO3A+/-),鉴定获得186 bp条带的为纯合型小鼠(FOXO3A-/-),鉴定获得100 bp条带的为野生型小鼠(FOXO3A+/+,WT);采用流式细胞术对鉴定出的野生型和纯合型小鼠进行骨髓细胞分型分析。结果 FOXO3A基因敲除小鼠已成功繁殖鉴定,子代基因敲除纯合型小鼠与杂合型小鼠长势正常,与野生型FVB/N小鼠相比外观、生长发育未见明显差异;已得到一定数量的纯合型小鼠用于后续实验;骨髓细胞计数结果显示,FOXO3A基因敲除纯合型小鼠骨髓有核细胞计数与野生型小鼠相比[(6. 167±1. 424) vs.(10±1. 732)],未见明显差异(P=0. 1625);流式分析结果显示:FOXO3A基因敲除小鼠骨髓中造血祖细胞(lin-scal-1~-ckit~+,HPC)比例明显升高(P<0. 05),造血干细胞(lin-scal-1~+ckit~+,HSC)比例没有明显差异;造血干细胞和造血祖细胞数目未发现有明显差异(P>0. 05)。结论经基因型鉴定已成功获得FOXO3A基因敲除小鼠。初步探讨FOXO3A基因敲除对小鼠骨髓细胞计数及分型未见明显影响,FOXO3A基因敲除对小鼠造血细胞的影响需要进一步的实验加以证明。
Objective To breed and identify FOXO3A gene knockout mice,hybrid mice were bred forconservation and homozygous mice were used for experiments. The effect of FOXO3A gene knockout on the hematopoietic cell phenotype in mice was preliminarily analyzed,laying the foundation for subsequent studies on the regulation of the FOXO3A gene during hematopoietic cell injury. Methods Hybrid female mice were mated with wild type male mice.Female and male hybrid mice were selected and bred,and homozygous mice were bred using one male and one or two females per cage. Pedicle marking and tail snips of neonatal mice were used to extract genomic DNA,and PCR was used for genotype identification. Heterozygous mice( FOXO3A+/-) with 100/186 bp bands,homozygous mice( FOXO3A-/-) with186 bp bands,and wild type mice( FOXO3A+/+,WT) with 100 bp bands were identified. Wild type and homozygous mice were used for bone marrow cell typing by flow cytometry. Results FOXO3A gene knockout mice were successfully bred and identified. Homozygous and heterozygous mice from the daughters of gene knockout mice had normal growth,with no obvious difference in appearance or development compared with wild type FVB/N mice. A number of homozygous mice were obtained for subsequent experiments. The bone marrow cell count of FOXO3A knockout homozygous mice contained similar numbers of nucleated cells compared with wild type mice( 6. 167±1. 424 vs 10± 1. 732; P = 0. 1625). The results of flow analysis showed that the proportion of hematopoietic progenitor cells( Lin-Scal-1~-c-kit~+,HPC) in the bone marrow of FOXO3A knockout mice was significantly increased( P<0. 05) compared with the wild type mice. In addition,there was no significant difference in the proportion of hematopoietic stem cells( Lin-Scal-1~+c-ki~t+,HSC),or number of hematopoietic stem cells and hematopoietic progenitor cells( P> 0. 05). Conclusions FOXO3A gene knockout mice were successfully obtained by genotype identification. Our preliminarily study demonstrated that FOXO3A gene knockout had no significant effect on the bone marrow cell count or typing in mice. Future studies will investigate the effect of FOXO3A gene knockout on hematopoietic cells in mice.
Objective To breed and identify FOXO3A gene knockout mice,hybrid mice were bred forconservation and homozygous mice were used for experiments. The effect of FOXO3A gene knockout on the hematopoietic cell phenotype in mice was preliminarily analyzed,laying the foundation for subsequent studies on the regulation of the FOXO3A gene during hematopoietic cell injury. Methods Hybrid female mice were mated with wild type male mice.Female and male hybrid mice were selected and bred,and homozygous mice were bred using one male and one or two females per cage. Pedicle marking and tail snips of neonatal mice were used to extract genomic DNA,and PCR was used for genotype identification. Heterozygous mice( FOXO3A+/-) with 100/186 bp bands,homozygous mice( FOXO3A-/-) with186 bp bands,and wild type mice( FOXO3A+/+,WT) with 100 bp bands were identified. Wild type and homozygous mice were used for bone marrow cell typing by flow cytometry. Results FOXO3A gene knockout mice were successfully bred and identified. Homozygous and heterozygous mice from the daughters of gene knockout mice had normal growth,with no obvious difference in appearance or development compared with wild type FVB/N mice. A number of homozygous mice were obtained for subsequent experiments. The bone marrow cell count of FOXO3A knockout homozygous mice contained similar numbers of nucleated cells compared with wild type mice( 6. 167±1. 424 vs 10± 1. 732; P = 0. 1625). The results of flow analysis showed that the proportion of hematopoietic progenitor cells( Lin-Scal-1~-c-kit~+,HPC) in the bone marrow of FOXO3A knockout mice was significantly increased( P<0. 05) compared with the wild type mice. In addition,there was no significant difference in the proportion of hematopoietic stem cells( Lin-Scal-1~+c-ki~t+,HSC),or number of hematopoietic stem cells and hematopoietic progenitor cells( P> 0. 05). Conclusions FOXO3A gene knockout mice were successfully obtained by genotype identification. Our preliminarily study demonstrated that FOXO3A gene knockout had no significant effect on the bone marrow cell count or typing in mice. Future studies will investigate the effect of FOXO3A gene knockout on hematopoietic cells in mice.
引文
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[14] Warr MR,Binnewies M,Flach J,et al. FOXO3A directs a protective autophagy program in haematopoietic stem cells[J].Nature,2013,494(7437):323-327.
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[2] Adamowicz M,Vermezovic J,d’Adda di Fagagna F. NOTCH1inhibits activation of ATM by impairing the formation of an ATMFOXO3a-KAT5/Tip60 complex[J]. Cell Rep,2016,16(8):2068-2076.
[3] Kops GJ, Dansen TB, Polderman PE, et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress[J]. Nature,2002,419(6904):316-321.
[4] Tothova Z,Kollipara R,Huntly BJ,et al. Fox Os are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress[J]. Cell,2007,128(2):325-339.
[5] Li X,Zhang T,Wilson A,et al. Transcriptional profiling of Foxo3a and Fancd2 regulated genes in mouse hematopoietic stem cells[J]. Genom Data,2015,4:148-149.
[6] Warr MR,Binnewies M,Flach J,et al. FOXO3A directs a protective autophagy program in haematopoietic stem cells[J].Nature,2013,494(7437):323-327.
[7] Miyamoto K,Miyamoto T,Kato R,et al. Fox O3a regulates hematopoietic homeostasis through a negative feedback pathway in conditions of stress or aging[J]. Blood,2008,112(12):4485-4493.
[8] Shao L,Feng W,Li H,et al. Total body irradiation causes longterm mouse BM injury via induction of HSC premature senescence in an Ink4a-and Arf-independent manner[J]. Blood,2014,123(20):3105-3115.
[9] Rehman A,Kim Y,Kim H,et al. FOXO3a expression is associated with lymph node metastasis and poor disease-free survival in triple-negative breast cancer[J]. J Clin Pathol,2018,71(9):806-813.
[10] Yu H1, Fellows A, Foote K, et al. FOXO3a(forkhead transcription factor O subfamily member 3a)links vascular smooth muscle cell apoptosis,matrix breakdown,atherosclerosis,and vascular remodeling through a novel pathway involving MMP13(matrix metalloproteinase 13)[J]. Arterioscler Thromb Vasc Biol,2018,38(3):555-565.
[11] She DT,Wong LJ,Baik SH,et al. SIRT2 inhibition confers neuroprotection by downregulation of FOXO3a and MAPK signaling pathways in ischemic stroke[J]. Mol Neurobiol,2018,55(12):9188-9203.
[12] Miyamoto,Araki,Y. Kiyomi,et al. Foxo3a is essential for maintenance of the hematopoietic stem cell pool[J]. Cell Stem Cell,2007,1(1):101-112.
[13] Tothova Z,Kollipara R,Huntly BJ,et al. Fox Os are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress[J]. Cell,2007,128(2):325-339.
[14] Warr MR,Binnewies M,Flach J,et al. FOXO3A directs a protective autophagy program in haematopoietic stem cells[J].Nature,2013,494(7437):323-327.
[15] Rossi DJ,Jamieson CH,Weissman IL. Stems cells and the pathways to aging and cancer[J]. Cell,2008,132(4):681-696.