A method of reprogramming fibroblasts into sertoli cells in vitro and its application
A technology of fibroblasts and fibroblasts, applied in the field of cell culture, can solve the problem of unrealistic large-scale acquisition of Sertoli cells
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Embodiment 1
[0092] Embodiment 1, construction contains the fibroblast cell line of AMH:GFP reporter system
[0093] The fibroblast cell line is dH1 fibroblast cell line or human lung fibroblast cell line.
[0094] 1. Construction of AMH:GFP reporting system
[0095] 1. The inventors of the present invention cloned the promoter A of the AMH gene from human genomic DNA. The promoter A of the AMH gene is about 1.6kb in length, and its nucleotide sequence is shown in sequence 1 in the sequence listing.
[0096] 2. After step 1 is completed, the gene encoding the fluorescent protein eGFP (i.e. the eGFP gene) is connected to the 3' end of the promoter A of the AMH gene to obtain the AMH:GFP reporter system. In the AMH:GFP reporter system, the expression of the fluorescent protein eGFP is driven by the promoter A of the AMH gene.
[0097] AMH (GeneID: 268) is a marker protein specifically expressed in the early stage of Sertoli cell development. If Sertoli cells are formed in cells introduce...
Embodiment 2
[0107] Example 2. In vitro reprogramming of human fibroblasts into AMH:GFP+Sertoli cells (hereinafter referred to as hiSCs)
[0108] Schematic flow chart of reprogramming human fibroblasts into hiSCs in vitro figure 2 .
[0109] 1. In vitro reprogramming of dH1 fibroblasts into hiSCs (dH1)
[0110] 1. Preparation of recombinant lentivirus
[0111] (1) Insert the NR5A1 gene into the recognition site of the restriction endonuclease EcoRI of the pENTR / 1A plasmid to obtain the vector pENTR / 1A-NR5A1. The GATA4 gene was inserted into the recognition site of the restriction endonuclease EcoRI of the pENTR / 1A plasmid to obtain the vector pENTR / 1A-GATA4. The WT1 gene was inserted between the recognition sites of the restriction endonucleases NotI and AscI of the pENTR / D-topo plasmid to obtain the vector pENTR / D-topo-WT1. The SOX9 gene was inserted between the recognition sites of the restriction endonucleases NotI and AscI of the pENTR / D-topo plasmid to obtain the vector pENTR / D-t...
Embodiment 3
[0137] Characteristics of human-derived fibroblasts reprogrammed into hiSCs in vitro in Example 3 and Example 2
[0138] 1. Analysis of Transcript Mapping
[0139] RNA sequencing was used to compare the transcriptional profiles of the tested cells (2F-hiSCs(dH1), 5F-hiSCs(dH1), 2F-hiSCs(HPF), 5F-hiSCs(HPF), adult Sertoli cells, or dH1-2K7), Then GO analysis.
[0140] Part of the transcript map see Figure 4 (both dH1-2K7-1 and dH1-2K7-2 are dH1-2K7, as a control; hiSCs-1 and hiSCs-2 are both 5F-hiSCs (dH1), aSCs-1 and aSCs-2 are both adult Sertoli cells) .
[0141] For some GO analysis results, see Figure 5 (dH1-2K7-1 and dH1-2K7-2 are both dH1-2K7, as a control; 2F-hiSCs(dH1)-1 and 2F-hiSCs(dH1)-2 are both 2F-hiSCs(dH1), 5F-hiSCs (HPF)-1 is 5F-hiSCs(HPF), 5F-hiSCs(dH1)-1 and 5F-hiSCs(dH1)-2 are 5F-hiSCs(dH1), aSCs-1 and aSCs-2 are adult Sertoli cell). Overall downregulated genes in 2F-hiSCs(HPF), 5F-hiSCs(HPF), 2F-hiSCs(dH1), 5F-hiSCs(dH1) and adult Sertoli cells comp...
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