Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for constructing a multi-gene transfected Tetrahymena thermophila cell line and its application

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

Active Publication Date: 2018-02-09
TAIYUAN UNIV OF TECH +1
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for constructing a multi-gene transfected Tetrahymena thermophila cell line and its application
  • A method for constructing a multi-gene transfected Tetrahymena thermophila cell line and its application
  • A method for constructing a multi-gene transfected Tetrahymena thermophila cell line and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Construction of plasmid pBsr

[0067] 1. PCR amplification of Bsr gene cassette sequence. The upstream and downstream primers are: 5′ GCGGCCGC GCATCTGGTGAGATATCTTCAAAGT-3' (the underlined part is the Not I restriction 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, preserved in our laboratory), the amplification conditions are: 94℃3min; 94℃30s, 51℃30s, 68℃1min10s, 30 cycles; 68℃5min. 1.0% agarose gel electrophoresis analysis of the amplified product is about 1.1 kb, a large amount of amplification and recovery of the target band (refer to BioFlux's DNA gel recovery kit manual for the gel recovery process), and sequencing to identify the Bsr gene cassette sequence. See attached for electrophoresis results figure 1 B.

[0068] 2. Digest the Bsr gene cassette and pNeo4 separately. (1) The Bsr gene cassette sequence was di...

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 the homologous recombination guided by the pNeo4-RAN1 recombinant plasmid, thereby achieving the purpose of knocking down the wild-type RAN1 gene. See attached for homologous recombination strategy figure 2 A, see attached diagram for construction image 3 .

[0072] 1. PCR amplification of 5'homologous sequence: 5'flanking sequence of RAN1 gene. The upstream and downstream primers are: 5′- GAGCTC ATTTACAATTATGCCATCTTCAT-3' (the underlined part is the Sac I restriction site), 5'- GCGGCCGC TTGTCTTCTGCCTTAACACACTA-3' (the underlined part is the Not I restriction site), the template is Tetrahymena genomic DNA, the amplification conditions are: 95℃ 3min; 95℃ 30s, 48℃ 30s, 68℃ 1 min, 30 Cycle; 68°C for 5 min. 1.0% agarose gel electrophoresis analysis of the amplified product is about 1.1 kb...

Embodiment 3

[0075] Example 3. Transfection of wild-type Tetrahymena with gene targeting recombinant plasmid pNeo4-RAN1 and screening and identification

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

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

[0078] (2) Cultivation of Tetrahymena. Take the wild type (Wild Type, WT) Tetrahymena thermophila CU428 and B2086 of different mating types that grow well and inoculate them in 50 mL SPP liquid medium (SPP liquid medium preparation method: use sterilized ultrapure water) 10× storage mother liquor, which contains 1% tryptone, 3‰ EDTA, 0.1% yeast extract, 1% prion protein, 0.2% glucose, autoclaved at 120℃ for 20 min, stored at -20℃, working concentration is 1×) , Make the initial concentration 0.1~0.5×10 4 cells / mL, incubate...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 present invention belongs to the field of biotechnology, and specifically relates to a method for constructing a multi-gene transfected Tetrahymena thermophila cell line, and the application of the method in the combined study of multiple genes of Tetrahymena. Background technique [0002] Tetrahymena thermophila (Tetrahymena thermophila) is a single-celled eukaryote, belonging to ciliated protozoa. It concentrates all the functions necessary to maintain life and extend the offspring in a single cell. It is an ideal model system for studying protein functions. As a biological model for studying the structure and function of certain human diseases-related genes, 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. the study. In recent years, with the deepening of genetics research, peopl...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/11C12N15/66
Inventor 梁海霞许静王伟
Owner TAIYUAN UNIV OF TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com