A transgenic rabbit carrying enhanced green fluorescent protein marker and its construction method

A technology of green fluorescent protein and green fluorescence, applied in the field of transgenic rabbits carrying enhanced green fluorescent protein markers and its construction, can solve the problems of affecting the early development of embryos, reducing the birth rate of transgenic animals, and the inability to obtain transgenic animals, etc., to achieve fluorescence Strong strength and photostability, shortened promoter sequence, and improved birth rate

Active Publication Date: 2019-07-12
NANJING NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

GFP transgenic rabbits were produced in France in 2000, but so far there has been no report of producing EGFP transgenic rabbits
Compared with GFP, EGFP has enhanced fluorescence intensity and photostability, and is more suitable for in vivo labeling; but strong EGFP expression will affect the early development of embryos, reduce the birth rate of transgenic animals, and even fail to obtain transgenic animals
Therefore, the construction of enhanced green fluorescent protein transgenic animals remains to be studied

Method used

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  • A transgenic rabbit carrying enhanced green fluorescent protein marker and its construction method
  • A transgenic rabbit carrying enhanced green fluorescent protein marker and its construction method
  • A transgenic rabbit carrying enhanced green fluorescent protein marker and its construction method

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Experimental program
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Embodiment 1

[0026] (1) Preparation and optimization of EGFP expression sequence

[0027] The structure of pCAG-GFP plasmid (Addgene, http: / / www.addgene.org / 11150 / ) is as follows figure 1 As shown, it includes a composite promoter sequence composed of CMV enhancer, chicken β promoter protein and CAG promoter, EGFP coding cDNA, and rabbit myosin polyA sequence; the pCAG-GFP plasmid was amplified in Escherichia coli and extracted in large quantities.

[0028] To compare the effect of the promoter sequence on embryonic development, two expression constructs were prepared. Conventional promoter expression components (hereinafter referred to as conventional components): pCAG-GFP plasmid was double digested with SpeI+hindIII, and a 3.1kb fragment was recovered, including a composite promoter sequence composed of CMV enhancer, chicken β promoter protein and CAG promoter, EGFP The coding cDNA, rabbit myosin polyA sequence. Optimized promoter expression component (hereinafter referred to as optim...

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Abstract

The invention relates to a transgenic rabbit with an enhanced green fluorescent protein gene marker, and a construction method thereof. EGFP (Enhanced Green Fluorescent Protein) expressed by the transgenic rabbit marks all cells of a rabbit individual; green fluorescent is emitted under the excitation of blue light, and a prominent genetic marker sample is used for distinguishing a non-marker sample. The construction method comprises the following steps: introducing a recombinant EGFP coding gene optimized by a promoter into a ribbit fertilized ovum to obtain an EGFP transgenic rabbit; carrying out EGFP gene detection or green fluorescent detection on a baby rabbit, wherein a positive result shows that the baby rabbit is a founder; carrying out hybridization on the founder and a normal rabbit to obtain an EGFP transgenic rabbit strain with genetic stability, and irradiating green fluorescent from rabbit tissues and culture cells subjected to line establishment. A promoter sequence is optimized, the EGFP is applied to manufacture the transgenic rabbit, and the rabbit marked by the EGFP can provide a visually visible biomarker sample from a macroscopic (individual) level to a microcosmic (cell) level.

Description

technical field [0001] The invention relates to a transgenic rabbit carrying an enhanced green fluorescent protein marker and a construction method thereof, in particular to constructing an enhanced green fluorescent protein transgenic rabbit through microinjection, and expressing an enhanced green fluorescent protein labeled rabbit individual All cells emit green fluorescence under excitation conditions of blue light. Background technique [0002] Green fluorescent protein (green fluorescent protein, GFP) is a tracer widely used in life science and medical research today. It is encoded by the GFP gene and emits green fluorescence when excited by light in the blue wavelength range. The optical imaging technology based on GFP enables people to directly observe the rich and colorful life phenomena from microcosmic to macrocosmic levels. [0003] GFP consists of 238 amino acids and has a molecular weight of 26.9kDa. It was originally isolated from Aequorea victoria and emits g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/89A01K67/027
CPCA01K67/0278A01K2227/107A01K2267/0393C12N15/89
Inventor 杜福良安礼友杨澜
Owner NANJING NORMAL UNIVERSITY
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