Process for preparing prion protein gene-free domestic animals

A gene knockout and prion protein technology, applied in the field of bioengineering

Inactive Publication Date: 2011-09-28
SHANGHAI GENON BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, there are no reports of PRNP gene mono-allelic or bi-allelic knockout goats in the prior art

Method used

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  • Process for preparing prion protein gene-free domestic animals
  • Process for preparing prion protein gene-free domestic animals
  • Process for preparing prion protein gene-free domestic animals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cloning of Goat Prion Gene and Construction of Goat Prion Gene Targeting Vector

[0055] 1. Culture and sex identification of primary goat fetal fibroblasts

[0056] Skin or ear tissue of 35-day-old goat fetuses was obtained, and then digested with a digestion solution containing 0.25% trypsin and 1 mM EDTA in a water bath at 37°C for about 15 minutes.

[0057] The digested single cell suspension was centrifuged, the supernatant was removed, and the cell pellet was resuspended in a solution containing 2mM glutamine (GIBCO), 1mM sodium pyruvate (SIGMA), and 1× non-essential amino acids (SIGMA). , 2ng / ml of basic fibroblast growth factor (bFGF, SIGMA), 10% fetal bovine serum (HyClone), 100units / ml of penicillin, 100μg / ml of streptomycin (SIGMA) in Glasgow minimal medium (GMEM) , SIGMA), the cell suspension was finally divided into culture flasks, cultured in a carbon dioxide incubator for several days, and frozen when the cell density reached about 80%.

[0058] The sex...

Embodiment 2

[0067] Knockout of goat PRNP gene using GTPrP targeting vector

[0068] After the vector GTPrP was linearized with SalI, it was transformed into the third passage GFF88 fetal fibroblast cell line by electrotransfection. Approximately 500 million fetal fibroblasts were mixed with 10 μg of linearized GTPRP vector, transferred to a 0.4 cm electroporation cuvette (Bio-Rad), and subjected to 220v, 950 μF by electroporation Gene pulserII (Bio-Rad). Pulsed, cells were then split into several dishes containing GMEM medium. After 48 hours G418 was added to a final concentration of 250 μg / ml.

[0069] About 10 days later, cell clones can be seen. The well-grown cell clones were picked out and transferred to the wells of a 96-well plate. When reaching confluence, some cells were isolated for PCR identification, and the remaining cells were further passaged until enough cells were obtained for cryopreservation and extraction of genomic DNA for identification by Southern hybridization, ...

Embodiment 3

[0087] Preparation of PRNP+ / - goats by nuclear transfer of goat PRNP+ / - cells

[0088] In the research of the present inventors, the donor oocytes, temporary host mothers and recipients for nuclear transfer were all from Saanen dairy goats.

[0089] Simultaneous estrus of goats: female goats were injected with 0.1 mg of prostaglandin-Cl (PG, Shanghai Institute of Family Planning Science), and 25 μg of luteinizing hormone-releasing hormone (LHRH) after 24 hours. Simultaneous estrus.

[0090] Superovulation in female goats: intramuscular injection of follicle stimulating hormone (FSH) twice a day for 3 consecutive days, the total dose of FSH per goat is 240IU, and PG is injected once on the third day, 24 One LHRH injection hours later.

[0091] Recovery of oocytes: Surgical recovery within 24-30 hours after LRH injection, oocytes were collected after flushing the fallopian tubes with F10 (GIBCO) medium, and 0.2% hyaluronidase was used to remove adhering granulosa cells, and th...

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PUM

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Abstract

The present invention discloses a preparation method for prion protein gene knockout domestic animals. The process is characterized by: knocking off the prion protein gene of domestic animals on a cellular level; and preparing prion protein gene knockout domestic animals through somatic cell cloning method. The function of the prion protein of domestic animals is inactivated. The functional prion protein is not expressed, thus the domestic animals can resist to relative disease of prion protein, such as scrapie, bovine spongiform encephalopathy (mad cow disease) and the like.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular, the invention relates to a method for preparing prion gene knockout livestock. Background technique [0002] Prion diseases, also known as Transmissible Spongiform Encephalopathies (TSE), are a class of fatal central nervous system degenerative diseases. The main features of the disease are neuronal cell death, vacuolation of the brain, and concomitant astrogliosis, which lead to movement disorders, dementia, and ultimately the death of diseased animals. The most familiar Ruan protein diseases are scrapie (scrapie) in sheep and goats, Bovine Spongiform Encephalopathy (BSE, commonly known as "mad cow disease") in cattle, and Kuru, Creutzfeldt-Jakobdisease (CJD), Gerstmann-Straussler-Sheinker disease (GSS), fatal familial insomnia (FFI), new Variant Creutzfeldt-Jakobdisease (vCJD) et al. (Prusiner SB: Priions. Proc Natl Acad Sci USA 1998, 95: 13363-13383). [0003] There is now a lot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/85A01K67/027
CPCY02A40/70
Inventor 成国祥陈建泉俞国华刘思国
Owner SHANGHAI GENON BIOENG
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