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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: 2007-05-30
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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Abstract

The invention discloses a making method of knocked livestock of protein gene, which is characterized by the following: losing activity for protein gene; making livestock possess relative disease of protein.

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 encephalopathy (Transmissible Spongiform Encephalopathies, TSE), is a kind of fatal central nervous system degenerative diseases. The main hallmarks of the disease are neuronal death, vacuolation of the brain, and concomitant astrogliosis, which lead to movement disorders, dementia, and ultimately death in the affected animals. The familiar Ruan protein diseases mainly include scrapie in sheep and goats, bovine spongiform encephalopathy (Bovine Spongiform Encephalopathy, BSE, commonly known as "mad cow disease") in cattle, and Kuru's disease (Kuru), Creutzfeldt-Jakobdisease (CJD), Gerstmann-Straussler-Sheinker disease (GSS), fatal familial insomnia (FFI) Jakob disease (Variant Creutzfeldt-Jakobdisease, vCJD) etc. (Prusine...

Claims

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Application Information

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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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