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Construction method and use of polygene-transfected tetrahymena thermophila cell strain

A technology of Tetrahymena thermophila and a construction method, which is applied in the application of the method in the multi-gene joint research of Tetrahymena thermophila, and the construction of a multi-gene transfected Tetrahymena thermophila cell line, which can solve problems such as inability to survive.

Active Publication Date: 2014-11-05
TAIYUAN UNIV OF TECH +1
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Problems solved by technology

Therefore, for the genes necessary for the nuclear differentiation and development of the sexual reproductive cells in the conjugative stage, the nuclear development will terminate, and the paired cells will separate and degrade; for the genes necessary for the growth of the vegetative reproductive cells, the offspring cells formed by the paired cells , will not survive due to lethal phenotype due to loss of essential genes in large nuclei

Method used

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  • Construction method and use of polygene-transfected tetrahymena thermophila cell strain
  • Construction method and use of polygene-transfected tetrahymena thermophila cell strain
  • Construction method and use of polygene-transfected tetrahymena thermophila cell strain

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Experimental program
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Embodiment 1

[0066] Embodiment 1, the construction of plasmid pBsr

[0067] 1. PCR amplification of the Bsr gene cassette sequence. The upstream and downstream primers are: 5' GCGGCCGC GCATCTGGTGAGATATCTTCAAAGT-3' (the underlined part is the Not I restriction enzyme site), 5' CTCGAG ATCATGATGGAAAATAAAACCATGCA-3′ (the underlined part is the Xho1 I restriction site), the template is pMNBL plasmid (gifted by Dr. Martin A. Gorovsky, University of Rochester, and kept in our laboratory), and the amplification conditions are: 94°C for 3min; 94°C for 30s, 51°C for 30s, 68°C for 1min10s, 30 cycles; 68°C for 5min. 1.0% agarose gel electrophoresis analysis of the amplified product was about 1.1 kb, a large number of amplified and recovered target bands (for the gel recovery process, refer to the instructions of BioFlux DNA gel recovery kit), sequenced and identified, and the sequence of the Bsr gene cassette was obtained. The results of electrophoresis are attached figure 1 b.

[0068] 2. Dige...

Embodiment 2

[0070] Example 2, Construction of Gene Targeting Recombinant Plasmid pNeo4-RAN1

[0071] The Neo4 gene cassette partially replaces the wild-type RAN1 gene on the large nuclear chromosome through homologous recombination guided by the pNeo4-RAN1 recombinant plasmid, thereby achieving the purpose of knocking down the wild-type RAN1 gene. Homologous recombination strategy see attached figure 2 A, construction diagram see attached image 3 .

[0072] 1. PCR amplification of the 5' homologous sequence: the flanking sequence at the 5' end of the RAN1 gene. The upstream and downstream primers are: 5′- GAGCTC ATTTACAATTATGCCATCTTCAT-3' (the underlined part is the Sac I restriction enzyme site), 5'- GCGGCCGC TTGTCTTCTGCCTTAACACACTA-3′ (the underlined part is the Not I restriction enzyme site), the template is Tetrahymena genomic DNA, the amplification conditions are: 95°C for 3min; 95°C for 30s, 48°C for 30s, 68°C for 1 min, 30 samples Cycle; 68°C for 5min. 1.0% agarose gel el...

Embodiment 3

[0075] Example 3, Gene Targeting Recombinant Plasmid pNeo4-RAN1 Transfection of Wild-type Tetrahymena and Screening and Identification

[0076] 1. pNeo4-RAN1 transfected wild-type Tetrahymena cells.

[0077] (1) Linearization and enrichment of gene targeting recombinant plasmid pNeo4-RAN1. The plasmid pNeo4-RAN1 was digested with Sac I and Kpn I to make it linearized, and the linearized fragment was concentrated to a concentration of 1-1.5 μg / μL.

[0078] (2) Culture of Tetrahymena. Take well-grown wild-type (Wild Type, WT) Tetrahymena thermophila CU428 and B2086 of different mating types, and inoculate them in 50 mL SPP liquid medium (SPP liquid medium preparation method: prepare with sterilized ultrapure water 10× stock solution containing 1% tryptone, 3‰ EDTA, 0.1% yeast extract, 1% prion protein, 0.2% glucose, autoclaved at 120°C for 20 min, stored at -20°C, working concentration is 1×) , so that the initial concentration is 0.1~0.5×10 4 cells / mL, cultured at a constan...

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Abstract

The invention discloses a construction method and a use of a polygene-transfected tetrahymena thermophila cell strain. The construction method comprises the following steps of cloning a homologous sequence of a target gene A in a targeting vector pNeo4, transforming tetrahymena in a nutritional stage by a particle bombardment method, carrying out paromomycin resistance screening to obtain a large nuclear gene-genetically transformed positive cell strain a, cloning a homologous sequence of a target gene B in a targeting vector pBsr, transforming the nutritional-stage tetrahymena cell strain a by the particle bombardment method, carrying out paromomycin-blasticidin combined resistance screening to obtain a large nuclear gene-genetically transformed positive cell strain a / b. The construction method solves the problem that the existing polygene transfection technology has low efficiency, a complicated screening process and long time, overcomes the limitation that the existing technology does not produce an essential gene-less or -mutant cell strain, and can be used for researching the essential gene-loss or -mutation-caused influence on related gene expression and function. The construction method can be used for directional modification such as knockout, mutation or epitope tagging of two different target genes thereby realizing tetrahymena polygene combined application research.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for constructing a tetrahymena thermophilic cell line transfected with multiple genes, and the application of the method in the joint research of multiple genes of the tetrahymena. Background technique [0002] Tetrahymena thermophila (Tetrahymena thermophila) is a single-celled eukaryote belonging to the ciliated protozoa. It concentrates all the functions necessary to maintain life and continue offspring in a single cell, and is an ideal model system for studying protein functions. As a biological model for studying the structure and function of genes related to certain human diseases, a series of mature gene recombination and transformation technologies have been established in Tetrahymena. These technologies have successfully achieved gene functions through gene knockout, overexpression and mutation. Research. In recent years, with the deepening of g...

Claims

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Application Information

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IPC IPC(8): C12N1/11C12N15/66
Inventor 梁海霞许静王伟
Owner TAIYUAN UNIV OF TECH
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