构建与制备RhoA-GTPase 突变体慢病毒
李良平, 顾晶晶, 王斌, 李娟, 张磊, 张琳, 张璐
中国临床解剖学杂志 ›› 2011, Vol. 29 ›› Issue (5) : 541-545.
构建与制备RhoA-GTPase 突变体慢病毒
Construction and identification of RhoA-GTPase lentivirus
目的 构建携带绿色荧光蛋白的RhoA显性负效突变体（RhoAN19）和组成型活性突变体（RhoAL63）慢病毒。 方法 构建RhoAN19和RhoAL63慢病毒表达质粒，并以酶切及序列测定方法进行鉴定。利用ViraPowerTM 慢病毒表达系统包装制备RhoA突变体慢病毒上清，用其感染大鼠前皮质神经元，分别进行RhoA生物学活性检测、细胞转染效率鉴定与神经元形态学观察。 结果 构建的RhoAN19和RhoAL63慢病毒表达质粒经酶切与测序鉴定正确，包装的慢病毒滴度为1×106 TU/ ml。用制备的慢病毒上清感染原代培养的前皮质神经元,生物学活性检测结果显示RhoAN19慢病毒显著抑制溶血磷脂酸（LPA）诱导的RhoA活性的升高，而RhoAL63慢病毒感染神经元后RhoA活性显著升高。感染效率鉴定结果显示病毒上清可感染80%以上的前皮质神经元。形态学观察显示经慢病毒感染后的神经元其胞体与树突分支清晰可见。 结论 成功制备了RhoA突变体慢病毒，并成功实现了慢病毒感染前皮质神经元，为进一步研究Rho蛋白家族信号通路提供了研究工具。
Objective To construct lentiviruses carrying dominant negative mutant of RhoA-GTPase (RhoAN19) or the constitutive active mutant of RhoA-GTPase (RhoAL63) and expressing enhanced green fluorescent protein (EGFP) bicistronically. Methods The lentiviral expression plasmid Plenti6/v5- RhoAN19 and Plenti6/v5- RhoAL63 were constructed and identified by restriction enzyme digestion and DNA sequence analysis. The two plasmids were packaged using the ViraPowerTM lentiviral expression system to produce replication-incompetent lentiviruses RhoAL63 and RhoAN19, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different RhoA mutants were evaluated and the morphology of the transfected PFCs was observed. Results The results of DNA sequencing and restriction enzyme analysis demonstrated correct plasmid construction. The packaged lentiviral titer was 1×106 TU/ml. Analysis of RhoA biological activity showed that RhoAN19 lentivirus particles infection significantly inhibited lysophospatidic acid stimulated RhoA activity in the PFCs, while RhoAL63 lentivirus particles enhanced the RhoA activity. The transfection efficiency of these RhoA mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses. Conclusions The lentiviruses carrying RhoA dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs. Thus providing important support for the study of RhoA signaling.
RhoA-GTPase / 慢病毒 / 绿色荧光蛋白 / 生物学活性
RhoA-GTPase / Lentivirus / Green fluorescent protein / Biological activity
[1] Wiggin GR, Fawcett JP, Pawson T. Polarity proteins in axon specification and synaptogenesis
[J]. Dev Cell, 2005, 8(6): 803-816.
[2] Auer M, Hausott B, Klimaschewski L. Rho GTPases as regulators of morphological neuroplasticity
[J]. Annals of Anatomy, 2011, 193(4):259-66.
[3] Murakoshi H, Wang H, Yasuda R. Local, persistent activation of Rho GTPases during plasticity of single dendritic spines
[J]. Nature, 2011,472(7341): 100-104.
[4] Govek EE, Newey SE, Van Aelst L.The role of the Rho GTPases in neuronal development
[J].Genes Dev, 2005, 19(1): 1-49.
[5] Ohnami S, Endo M, Hirai S, et al. Role of RhoA in activity-dependent cortical axon branching
[J]. J Neurosci, 2008, 10;28(37):9117-21.
[6] Iden S., Collard JG. Crosstalk between small GTPases and polarity proteins in cell polarization
[J]. Nat Rev Mol Cell Biol, 2008, 9(11):846-59.
[7] Chilton JK. Molecular mechanisms of axon guidance
[J]. Dev Biol, 2006, 292(1):13-24.
[8] Shang X, Cancelas JA, Li L,et al. R-Ras and Rac GTPase crosstalk regulates hematopoietic progenitor cell migration, homing and mobilization
[J]. J Biol Chem , 2011,
[Epub ahead of print]
[9] Mulloy JC, Cancelas JA, Filippi MD, et al. Rho GTPases in hematopoiesis and hemopathies
[J]. Blood, 2010 ,115(5):936-947.
[10] Guo F, Velu CS, Grimes HL, et al. Rho GTPase Cdc42 is essential for B-lymphocyte development and activation
[J]. Blood, 2009, 114(14):2909-2916.
国家自然科学基金(30770821,81071120,30973129 )；教育部新世纪优秀人才支持计划（NCET-09-0088）；广东省自然科学基金重点项目（92510515000008）；广东省科技计划项目(2009A030200015)；教育部高等学校博士学科点专项基金(20094433120012); 教育部科学技术研究重点项目(20103388008)
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