Novel method for obtaining goat-induced multi-potent stem cells

Abstract

The invention discloses a novel method for obtaining goat-induced multi-potent stem cells. The novel method comprises the following steps: firstly, extracting total RNAs from a testicular tissue, a skin tissue and a small intestine tissue of a goat; performing reverse transcription to obtain a cDNA segment; and with the cDNA segment as a template, cloning to obtain coding sequences of Oct4, Sox2, K1f4 and c-Myc multifunctional transcription factors of the goat. Fetal fibroblast cells of the goat and a mouse are obtained by utilizing a trypsin digestion method, feed-layer cells and a cell culture solution are prepared. mRNA of each multifunctional transcription factor obtained by utilizing an in-vitro transcription test is mediated by lipidosome 2000 to transfect the goat fetal fibroblast cells, so that goat iPS cells are prepared. A cell colony which is flatly cloned, dense, large in nuclear-cytoplasmic ratio, clear in cloning boundary and strong in light refraction is judged as a goat iPS cell colony. The goat iPS cell colony is picked in a monoclonal mode, digested by pancreatic enzyme and independently inoculated to a novel culture pore plate inoculated with a feeding layer to be further cultured to obtain a goat iPS cell line.

Description

Technical field:

[0001] The invention relates to the fields of stem cell engineering, preservation of rare species, tissue and cell engineering and the like. technical background:

[0002] The introduction of pluripotent transcription factors in the production of goat iPS cells requires a suitable delivery system, and the efficiency and safety of the delivery system introduction method is the research focus of iPS cell preparation technology. The current research methods of delivery systems are mainly divided into four categories: gene insertion non-deletable type, including retrovirus, constitutive and inducible lentivirus, etc. The method is to infect 293 or 293T cells with viral vectors connected with pluripotent transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 to prepare virus particles, and then infect target cells to prepare iPS cells. The vector cannot be deleted in the target cell and can be randomly integrated in the target cell; gene insertion can b...

Examples

Embodiment Construction

[0018] 1. Cloning of pluripotency transcription factors

[0019] ①Total RNA extraction

[0020] The total RNA of goat testis tissue, skin tissue and small intestine tissue was extracted respectively by RNA extraction kit, and the extraction steps were as follows:

[0021] (1) Add 50-100mg of goat testicular tissue, skin tissue and small intestine tissue samples to the mortar treated with DEPC water, add liquid nitrogen and quickly grind to powder, put them into 1.5mL EP tubes, and quickly Add 1mL of Trizol, mix well and let stand at room temperature for 10 minutes;

[0022] (2) Centrifuge in a refrigerated centrifuge at 4°C and 12,000 rpm for 10 minutes, transfer the supernatant to a new EP tube, add 200 μL of chloroform and shake vigorously repeatedly for 15 seconds, place at 25°C for 3 minutes, and then centrifuge for 15 minutes with the same standard;

[0023] (3) Transfer the upper aqueous phase in the EP tube to another new tube, add 400 μL of isopropanol, mix well, lea...

Technical field:

[0001] The invention relates to the fields of stem cell engineering, preservation of rare species, tissue and cell engineering and the like. technical background:

[0002] The introduction of pluripotent transcription factors in the production of goat iPS cells requires a suitable delivery system, and the efficiency and safety of the delivery system introduction method is the research focus of iPS cell preparation technology. The current research methods of delivery systems are mainly divided into four categories: gene insertion non-deletable type, including retrovirus, constitutive and inducible lentivirus, etc. The method is to infect 293 or 293T cells with viral vectors connected with pluripotent transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 to prepare virus particles, and then infect target cells to prepare iPS cells. The vector cannot be deleted in the target cell and can be randomly integrated in the target cell; gene insertion can b...

Examples

Embodiment Construction

[0018] 1. Cloning of pluripotency transcription factors

[0019] ①Total RNA extraction

[0020] The total RNA of goat testis tissue, skin tissue and small intestine tissue was extracted respectively by RNA extraction kit, and the extraction steps were as follows:

[0021] (1) Add 50-100mg of goat testicular tissue, skin tissue and small intestine tissue samples to the mortar treated with DEPC water, add liquid nitrogen and quickly grind to powder, put them into 1.5mL EP tubes, and quickly Add 1mL of Trizol, mix well and let stand at room temperature for 10 minutes;

[0022] (2) Centrifuge in a refrigerated centrifuge at 4°C and 12,000 rpm for 10 minutes, transfer the supernatant to a new EP tube, add 200 μL of chloroform and shake vigorously repeatedly for 15 seconds, place at 25°C for 3 minutes, and then centrifuge for 15 minutes with the same standard;

[0023] (3) Transfer the upper aqueous phase in the EP tube to another new tube, add 400 μL of isopropanol, mix well, lea...

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