核定位信号肽在异种移植动物模型转人α1,2-岩藻糖苷转移酶基因小鼠制备中的作用

Nuclear localization signals enhance integration and expression of human α1, 2-fucosyltransferase gene in mice

目的探讨核定位信号肽(NLS)在转人α1,2-岩藻糖苷转移酶(HT)基因小鼠制备中的作用。方法将受精卵随机分为3组,采用显微注射的方法制备转基因小鼠。实验组:将NIS 转基因构件与人HT转基因构件同时导入细胞质;对照Ⅰ组:将人HT转基因构件导入细胞质;对照Ⅱ组:将人HT转基因构件导入细胞核。应用聚合酶链反应(PCR)、Southern杂交及逆转录 (RT)-PCR等方法检测人HT基因在G0代小鼠体内的整合与表达,应用流式细胞计数(FCM)检测转基因小鼠外周血单核细胞(PBMCs)表面H抗原与α-Gal抗原的表达。结果实验组与对照Ⅰ组 G0代小鼠的出生率33．8％(46／136)及35．4％(23／65)高于对照Ⅱ组10．2％(51／498)(P<0．01),实验组人HT基因的整合率(30．4％,14／46)高于对照Ⅰ组(0,0／23)与对照Ⅱ组(11．8％,6／51,P< 0．01),实验组人HT基因的表达率(19．6％,9／46)也高于对照Ⅰ组(0,0／23)和对照Ⅱ组(5．9％,3／ 51,P<0．01),人HT mRNA在小鼠心、肝、肾和骨骼肌组织中均表达阳性。转基因小鼠PBMCs表面H抗原表达阳性。表达强度为人的85％～190％,同时α-Gal抗原表达显著降低,为正常小鼠的 5％～12％。转基因小鼠已经稳定传3代。结论 NLS基因与目的基因细胞质共注射是制备转基因小鼠简便实用的新方法。

Objective To investigate the role of nuclear localization signals （NLS） in producing transgenic mice of human α1, 2-fucosyltransferase （HT） gene. Methods The zygotes were divided into three groups at random and transgenic mice were produced by microinjection. Experimental groups： human HT transgene construct mixed with NLS transgene construct were co-injected into murine zygote cytoplasm;Control Ⅰ group：Only human HT transgene construct was injected into cytoplasm; Control Ⅱ group：Human HT transgene construct was injected into cytonucleus. PCR and Southern-blotting were used to screen the positive transgenic mice. RT-PCR analysis was used to detect the level of human HT mRNA in transgenic mice. The expression of H antigen and α-Gal antigen in peripheral blood mononuclear cells （PBMCs） of transgenic mice was detected by flow cytometry （FCM）. Results Birthrate of experimental and control Ⅰ and Ⅱ groups was 33.8% （46/136）,35.4% （23/65）and 10.2% （51/498） respectively. Integation rate of human HT gene was significantly higher in the expremental groups （30.4%,20/46） than in control Ⅰ （0,0/23） and control Ⅱ groups （11.8%,6/51） （P〈0.01）.The expression rate of human HT gene was also significantly higher in experimental groups （ 19.6 %, 9/46 ） than in control Ⅰ （0, 0/23） and control Ⅱ groups （ 5.9 %, 3/51） （ P 〈 0.01）. The expression of human HT gene was positive in organs of transgenic mice including heart, liver, kidney and muscle. H antigen was positively expressed and α-Gal antigen was decreased in PBMCs of transgenic mice. The human HT gene was transferred to the third generation. Conclusion NLS could enhance integration and expression of human HT gene in mice. Cytoplasm co-injection of NLS transgene contruct and heterologous gene contruct is a new method of producing transgenic mice.