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Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors

A technology for pluripotent stem cells and transcription factors, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and cells modified by introducing foreign genetic material, which can solve the problem of increasing workload and difficulty, affecting the clinical application of ESCs cells, etc. problem, to achieve good pluripotency and avoid the effect of xenogeneic animal cell contamination

Inactive Publication Date: 2012-06-06
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of mouse embryonic fibroblasts in the ESCs culture system directly introduces xenogeneic animal cells, which directly affects the clinical application of ESCs cells, and also increases the workload and difficulty in the culture process

Method used

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  • Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors
  • Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors
  • Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Isolation and cultivation of bovine skin fibroblasts of embodiment 1

[0052] Adult bovine dermal fibroblasts (BDFs) were isolated from the ear margin skin of female Holstein cows. After the tissue was cleaned and sterilized, it was cut into small tissue pieces, and cultured in high-sugar DMEM (Gibco company product) containing 15%-20% fetal bovine serum (FBS) (Gibco company product) under conventional culture conditions. The culture solution was changed every 2-3 days. After 7-10 days, fibroblasts moved out from the edge of the tissue block, and after 12-16 days, a large number of fibroblasts were tightly arranged around the tissue block. A large number of adult bovine skin cells can be obtained by digestion and passage with the mixed digestive solution of trypsin and EDTA, see figure 1 . Newborn bull skin fibroblasts (male newborn bovine dermal fibroblasts, NDFs) come from Holstein breed newborn bull ear margin skin, see figure 2 , the method of isolation and cult...

Embodiment 2

[0053] Example 2 Cloning of key gene transcription factor for bovine maintenance of pluripotency and construction of its retroviral vector

[0054] Genital ridges were isolated from fetuses of 50-60-day-old Holstein cows, and the total RNA was extracted with TRIzol LS Reagent after tissue homogenization, and treated with DNaseI (RNase free) to remove genomic DNA contamination. cDNA was obtained by reverse transcription reaction. Then, using cDNA as a template, the complete sequences of the open reading frames (ORFs) of Oct4, Sox2, Nanog and Lin28 transcription factor genes were respectively amplified by PCR. The primer sequences used for gene cloning are shown in Table 1.

[0055] Table 1 Amplifies information of primers for RT-PCR amplification of four genes of bovine Oct4, Sox2, Nanog, and Lin28

[0056]

[0057]

[0058] Among them, the PCR amplification parameters of Oct4 gene are: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 sec, annealing at 60°...

Embodiment 3

[0064] Packaging and preparation of embodiment 3 retrovirus

[0065] PT67 packaging cells were cultured in DMEM medium according to conventional methods. When the cells reached 70%-80% confluence, liposomes were used to transfect the four retroviral vectors into the packaging cells PT67 cells, and the virus packaging was carried out to obtain Infectious retroviral particles. One hour before transfection, the DMEM medium was replaced with serum-free medium, and 4-8 μg of retroviral plasmid DNA and 10-20 μl of liposomes were added to 2 parts of 500 μl Opti-MEM-I+GlutaMAX-I (Gibco company product ) in the optimized transfection solution, mix gently, and incubate at room temperature, then gently mix the diluted plasmid DNA and liposomes, and incubate at room temperature. After 20 minutes, the mixture was added dropwise to the PT67 cell culture dish. Cells at 37°C, 5% CO 2 Cultivate for 4-6 hours under saturated humidity conditions. Afterwards, the transfection solution was rep...

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Abstract

The invention discloses a method for transfecting bovine somatic cells into inducted pluripotent stem cells (iPSCs) by adopting transcription factors, concretely comprising the following steps: 1) isolation and culture of bovine dermal fibroblasts; 2) cloning of bovine transcription factor genes maintaining pluripotency and construction of retroviral vectors of the transcription factor genes; 3) retrovirus packaging and preparation; and 4) infection of bovine dermal fibroblasts by retrovirus and acquisition of bovine inducted pluripotent stem cells. The invention first adopts the acellular human amniotic membranes as support to replace the mouse embryonic fibroblasts (MEFs) to maintain in vitro proliferation of the bovine iPSCs and keep the bovine iPSCs in undifferentiated pluripotent states for long time. The method which adopts gene inducing to generate the PSCs not only solves the technical problem that the PSCs of the animals such as cattle, sheep, pigs and other livestock are difficult to obtain but also avoids the problem of heterogeneous animal cell pollution caused by the feeder layer which applies the MEFs as the stem cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for transfecting bovine somatic cells into induced pluripotent stem cells by using transcription factors. Background technique [0002] In 2006, Japanese scholar Yamanaka [1] It was reported for the first time that mouse somatic cells were co-transfected with a combination of mouse transcription factors Oct4, Sox2, c-Myc, and Klf4, and mouse induced pluripotent stem cells (iPSCs) were obtained. Subsequently, a worldwide research upsurge of induced pluripotent stem cells was set off. Studies have been made on many aspects such as animal species, types and quantities of transcription factors, combinations of transcription factors, and foreign gene introduction methods. 2007Yamanaka [2] Using the same transcription factor to successfully obtain human iPSCs; in the same year, Yu (Yu Junying) and Thomson [3] et al. also obtained human iPS cells usi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 吕长荣陈冬梅辛晓玲窦忠英
Owner NORTHWEST A & F UNIV
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