一种优化的成年斑马鱼视网膜吗啡啉电转染方法
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摘要
通过吗啡啉(morpholino,MO)电转染敲降斑马鱼视网膜内目标基因表达是研究基因功能的有效手段,但现有方法存在局限性。本文旨在发展一种优化的成年斑马鱼视网膜MO电转染方法。优化要点是在电极内侧涂抹超声凝胶,并使用方形电极而不是圆形电极。结果显示,使用超声凝胶可有效减少电转染所致视网膜损伤并简化实验步骤,采用方形电极显著提高了MO电转染效率。用本方法敲降视网膜再生相关基因Ascl1a显著抑制了视网膜祖细胞的生成。本方法是对现有MO电转染方法的优化。
In vivo electroporation of morpholinos(MOs) into the retina of adult zebrafish is an efficient method to study gene function related to retinal disease and regeneration. However, the currently reported methods are complicated with low MO transfer efficiency and high probability to cause collateral damage. The present study was aimed to optimize the existing MO electroporation methods. Two major changes were made to MO electroporation procedure in zebrafish retina. One was to coat the inner side of the electrode with ultrasonic gel. The other was to replace the commonly used round electrode with novel rectangular one. The results showed that the use of ultrasonic gel reduced collateral damage caused by retinal electroporation and simplified the experimental procedure. The rectangular electrode significantly increased transfection efficiency of MO electroporation. In particular, knocking down the expression of Ascl1 a in the retina by using our method significantly inhibited the generation of retinal progenitor cells. These results suggest our method is the optimization of the current MO electroporation methods and may be a better alternative for relevant researchers.
In vivo electroporation of morpholinos(MOs) into the retina of adult zebrafish is an efficient method to study gene function related to retinal disease and regeneration. However, the currently reported methods are complicated with low MO transfer efficiency and high probability to cause collateral damage. The present study was aimed to optimize the existing MO electroporation methods. Two major changes were made to MO electroporation procedure in zebrafish retina. One was to coat the inner side of the electrode with ultrasonic gel. The other was to replace the commonly used round electrode with novel rectangular one. The results showed that the use of ultrasonic gel reduced collateral damage caused by retinal electroporation and simplified the experimental procedure. The rectangular electrode significantly increased transfection efficiency of MO electroporation. In particular, knocking down the expression of Ascl1 a in the retina by using our method significantly inhibited the generation of retinal progenitor cells. These results suggest our method is the optimization of the current MO electroporation methods and may be a better alternative for relevant researchers.
引文
1 Dooley K,Zon LI.Zebrafish:a model system for the study of human disease.Curr Opin Genet Dev 2000;10(3):252-256.
2 Glass AS,Dahm R.The zebrafish as a model organism for eye development.Ophthalmic Res 2004;36(1):4-24.
3 Fadool JM,Dowling JE.Zebrafish:a model system for the study of eye genetics.Prog Retin Eye Res 2008;27(1):89-110.
4 Goldman D.Müller glial cell reprogramming and retina regeneration.Nat Rev Neurosci 2014;15(7):431-442.
5 Lenkowski JR,Raymond PA.Müller glia:Stem cells for generation and regeneration of retinal neurons in teleost fish.Prog Retin Eye Res 2014;40:94-123.
6 Mokalled MH,Patra C,Dickson AL,Endo T,Stainier DY,Poss KD.Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.Science 2016;354(6312):630-634.
7 Thummel R,Enright JM,Kassen SC,Montgomery JE,Bailey TJ,Hyde DR.Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration.Exp Eye Res 2010;90(5):572-582.
8 Fausett BV,Gumerson JD,Goldman D.The proneural basic helix-loop-helix gene ascl1a is required for retina regeneration.J Neurosci 2008;28(5):1109-1117.
9 Thummel R,Bailey TJ,Hyde DR.In vivo electroporation of morpholinos into the adult zebrafish retina.J Vis Exp 2011;(58):e3603.
10 Zhang S,Mu Z,He C,Zhou M,Liu D,Zhao XF,Goldman D,Xu H.Antiviral drug ganciclovir is a potent inhibitor of the proliferation of Müller glia-derived progenitors during zebrafish retinal regeneration.Invest Ophthalmol Vis Sci2016;57(4):1991-2000.
11 Mu Z,Zhang S,He C,Hou H,Liu D,Hu N,Xu H.Expression of Sox C transcription factors during zebrafish retinal and optic nerve regeneration.Neurosci Bull 2017;33(1):53-61.
12 Wan J,Ramachandran R,Goldman D.HB-EGF is necessary and sufficient for Müller glia dedifferentiation and retina regeneration.Dev Cell 2012;22(2):334-347.
13 Ramachandran R,Fausett BV,Goldman D.Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent,let-7 micro RNA signalling pathway.Nat Cell Biol 2010;12(11):1101-1107.
2 Glass AS,Dahm R.The zebrafish as a model organism for eye development.Ophthalmic Res 2004;36(1):4-24.
3 Fadool JM,Dowling JE.Zebrafish:a model system for the study of eye genetics.Prog Retin Eye Res 2008;27(1):89-110.
4 Goldman D.Müller glial cell reprogramming and retina regeneration.Nat Rev Neurosci 2014;15(7):431-442.
5 Lenkowski JR,Raymond PA.Müller glia:Stem cells for generation and regeneration of retinal neurons in teleost fish.Prog Retin Eye Res 2014;40:94-123.
6 Mokalled MH,Patra C,Dickson AL,Endo T,Stainier DY,Poss KD.Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.Science 2016;354(6312):630-634.
7 Thummel R,Enright JM,Kassen SC,Montgomery JE,Bailey TJ,Hyde DR.Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during zebrafish photoreceptor regeneration.Exp Eye Res 2010;90(5):572-582.
8 Fausett BV,Gumerson JD,Goldman D.The proneural basic helix-loop-helix gene ascl1a is required for retina regeneration.J Neurosci 2008;28(5):1109-1117.
9 Thummel R,Bailey TJ,Hyde DR.In vivo electroporation of morpholinos into the adult zebrafish retina.J Vis Exp 2011;(58):e3603.
10 Zhang S,Mu Z,He C,Zhou M,Liu D,Zhao XF,Goldman D,Xu H.Antiviral drug ganciclovir is a potent inhibitor of the proliferation of Müller glia-derived progenitors during zebrafish retinal regeneration.Invest Ophthalmol Vis Sci2016;57(4):1991-2000.
11 Mu Z,Zhang S,He C,Hou H,Liu D,Hu N,Xu H.Expression of Sox C transcription factors during zebrafish retinal and optic nerve regeneration.Neurosci Bull 2017;33(1):53-61.
12 Wan J,Ramachandran R,Goldman D.HB-EGF is necessary and sufficient for Müller glia dedifferentiation and retina regeneration.Dev Cell 2012;22(2):334-347.
13 Ramachandran R,Fausett BV,Goldman D.Ascl1a regulates Müller glia dedifferentiation and retinal regeneration through a Lin-28-dependent,let-7 micro RNA signalling pathway.Nat Cell Biol 2010;12(11):1101-1107.