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A technology of ganglion cells and retina, applied in the field of biomedicine, can solve the problem of insufficient efficiency of retinal ganglion cell-like neurons and achieve high efficiency
Pending Publication Date: 2019-10-08
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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Although it has been reported that overexpression of Ascl1, Brn3b and Ngn2 can reprogram mouse fibroblasts into Brn3a-positive retinal ganglion cell-like cells (Neuroscience 2013,250:381-93), the method in this report obtained retinal ganglion cells The efficiency of the neurons is not high enough
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Embodiment 1
[0041] This embodiment provides a method for preparing retinal ganglion cells, the specific steps comprising:
[0042] Step 1. Preparation of lentiviral expression plasmid pSicoR-TetO-Ascl1-P2A-Islet1-T2A-Brn3b (pSicoR-TetO-ABI)
[0043] (1) Obtain the full-length cDNA of Ascl1 gene, Brn3b gene and Islet1 gene
[0044] 1) Total RNA was extracted from head tissues of mouse embryos on day 13.5, and reverse-transcribed into a cDNA library using reverse transcriptase Superscript III (ThermoFisher, Scientific).
[0045] 2) Using the cDNA library as a template and using the following SEQ ID No.1 and SEQ ID No.2 as primers, perform PCR amplification to obtain the full-length cDNA of the mouse Brn3b gene;
[0046] SEQ ID No.1: 5'-gaactgtacaatgatgatgatgtccctgaac-3',
[0047] SEQ ID No.2: 5'-ggttgaattcctaaatgccggcagagtatttc-3';
[0048] The PCR amplification system is: 1μl cDNA library, 1×Phusion reaction buffer, 200μM dNTP, 1μM forward primer SEQ ID No.1, 1μM reverse primer SEQ ID No....
Embodiment 2
[0088] This example is a modification of Example 1, and provides a method for preparing retinal ganglion cells. Compared with Example 1, the changes are that the exogenous Ascl1 gene, the exogenous Islet1 gene, and the exogenous Brn3b The genes are located on different plasmids. Preparation methods include:
[0090] (1) The lentiviral expression plasmid carrying exogenous Ascl1 gene directly used TetO-FUW-Ascl1, which was purchased from Addgeng, Cat. No. 27150.
[0091] (2) Construction of lentiviral expression plasmid pSicoR-TetO-Islet1
[0092] 1) Refer to Example 1 to obtain the full-length cDNA of Islet1 gene (Islet1-cDNA), wherein the amplification primers are replaced with:
[0093] SEQ ID No.10: 5'-ggttgctagccaccatgggagacatgggcgatcc-3',
[0094] SEQ ID No. 11: 5'-ggtatgtacatcatgcctcaataggactgg-3'.
[0095] 2) Cloning Islet1-cDNA into the ind...
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Abstract
The present invention provides a method for preparing retinal ganglion cells, which comprises the following steps of: obtaining a somatic cell; constructing lentiviral particles, transfecting the somatic cell with the lentiviral particles, integrating an Ascl1 gene, a Islet1 gene, and a Brn3b gene into the somatic cell chromosome; and performing induction culture to obtain the retinal ganglion cells. The retinal ganglion cells are obtained by integrating the exogenous Ascl1 gene, the Islet1 gene, and the Brn3b gene into the mouse fibroblast chromosome for overexpression preparation. Compared with the prior art, the present invention creatively selects a combination of the Ascl1 gene, the Brn3b gene and the Islet1 gene, the three transcription factors are overexpressed in the somatic cellsby lentivirus-mediated means, and more than 10% of the somatic cells can be transdifferentiated into Brn3a-positive, Tuj1-positive retinal ganglion cells in 7 days of induction culture, and the efficiency is high.
Description
technical field [0001] The invention belongs to the field of biomedicine, in particular to a preparation method of retinal ganglion cells. Background technique [0002] Since the Nobel Prize winner Shinya Yamanaka discovered that by overexpressing four transcription factors Oct4, Sox2, Klf4 and c-Myc, somatic cells can be reprogrammed into pluripotent stem cells (iPS), people have successively discovered that tissue-specific transcription factors can be reprogrammed by overexpressing Somatic cells can be directly reprogrammed into somatic cells of other lineages without going through the pluripotent stem cell stage (direct reprogramming of somatic cells). In 2010, the laboratory of Marius Wernig in the United States found that the simultaneous overexpression of Ascl1, Brn2 and Myt1l could efficiently and directly reprogram mouse fibroblasts into Tuj1-positive neurons (Nature 2010, 463: 1035-41). This discovery opens up a new, convenient and fast way to prepare donor cells f...
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