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Application of miRNA-19a (micro ribonucleic acid-19a) serving as target in regulation of mammal host cell proliferation

A miRNA-19a, 1.miRNA-19a technology, applied in the field of mammalian host cell remodeling and improvement, can solve other problems such as cell death, low proliferation rate, and apoptosis

Inactive Publication Date: 2013-11-20
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, CHO cells, like other mammalian cells, have some shortcomings that are not suitable for large-scale industrial cultivation, such as low proliferation rate, high requirements for culture environment, poor tolerance to various stress environments, and various unfavorable environments. Factors such as nutrient deficiency, accumulation of harmful metabolites, too high or too low dissolved oxygen, mechanical agitation, etc. can affect cell proliferation and even cause cell apoptosis, especially in serum-free medium, cells are more likely to grow slowly and undergo apoptosis
Another problem is that the growth of mammalian cells is not easy to control
If cells continue to maintain a high growth rate, they will soon reach the upper limit of the capacity of the bioreactor. Due to the weak contact inhibition mechanism of cells cultured in vitro, overcrowded cells will lead to density-dependent apoptosis. Initiation of death, the content of dead cells is released into the medium, which will further cause the death of other cells, and then stop the production process in advance

Method used

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  • Application of miRNA-19a (micro ribonucleic acid-19a) serving as target in regulation of mammal host cell proliferation
  • Application of miRNA-19a (micro ribonucleic acid-19a) serving as target in regulation of mammal host cell proliferation
  • Application of miRNA-19a (micro ribonucleic acid-19a) serving as target in regulation of mammal host cell proliferation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Analysis of the relationship between miRNA-19a expression level and cell growth rate

[0025] 1. Screening of cell clones with different growth rates

[0026] Twenty CHO cell clones were inoculated into 60mL glass spinner bottles, and each clone was inoculated in 3 bottles. Serum-free DMEM (Hyclone) culture medium was used for suspension culture at 37°C for 72 hours, and the cell growth rate was analyzed. The growth rate is calculated according to the following formula:

[0027]

[0028] In the formula, cell density 2 is the cell density at the end of the culture, cell density 1 is the inoculated cell density, and time 2-time 1 is the cell culture time.

[0029] The results showed that the growth rate of 20 clones ranged from 0.011 to 0.044hr -1 , of which 5 clones (H1~H5) were identified as having high growth rate (≥0.035hr -1 ), 5 clones (L1~L5) were identified as having low growth rate (≤0.020hr -1 ).

[0030] 2. microRNA microarray chip analysis

...

Embodiment 2

[0034] Example 2 Construction of miRNA-19a recombinant expression cell line

[0035] 1. Construction of miRNA-19a high expression cell line

[0036] According to the precursor sequence pre-miRNA-19a (SEQ ID No.6) of miRNA-19a, find the genome sequence region where the precursor sequence is located from the published hamster genome sequence through NCBI blast alignment, refer to pre-miRNA- The 5' and 3' ends of the 19a sequence were extended by about 100 bp (SEQ ID No.7), and the following primers were designed and synthesized: Forward primer pre-miRNA19a-F: 5'-gaccggtgcatctactgccctaagtgc-3' (SEQ ID No.8 ), reverse primer pre-miRNA19a-R: 5'-ggaattcctgcactataagcactttagtgc-3' (SEQ ID No.9). The total RNA of CHO cells extracted above was taken, reverse-transcribed into cDNA using a reverse transcription kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and operated according to the kit instructions. Then, using the obtained cDNA as a template, the pre-miRNA-19a sequence ...

Embodiment 3

[0043] Example 3 Prediction and verification of miRNA-19a target gene

[0044] 1. Prediction of miRNA-19a target genes

[0045] The miRNA target gene prediction software Target Scan Human6.2 was used to predict the target gene of miRNA-19a, and it was found that Cyclin D1 of human, mouse and other animals is the potential target gene of this miRNA. A comparative analysis of the Cyclin D1 cDNA sequences from 3 species of hamster (EF524275.1), human (NM_053056.2), and mouse (X75207.1) registered in Genbank found that the amino acid sequence and corresponding codon sequence of hamster Cyclin D1 were similar to those of human, Rats are exactly the same. The genome sequence of hamster (JH000254.1) was analyzed again, and it was found that the 3'-UTR of hamster Cyclin D1 mRNA contained the paired region of hamster miRNA-19a seed sequence. Therefore, it is predicted that Cyclin D1 may be the target gene of miRNA-19a in CHO cells.

[0046] 2. Verification of miRNA-19a target genes ...

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Abstract

The invention discloses an application of miRNA-19a (micro ribonucleic acid-19a) serving as a target in regulation of mammal host cell proliferation. A genetic engineering technology is used for inhibiting or promoting the expression quantity of the miRNA-19a in a mammal host cell, so that the expression quantity of cell cycle protein Cyclin D1 closely related to cell proliferation can be indirectly promoted or inhibited, and further the cell proliferation is promoted or inhibited, thus the purpose of regulating the mammal host cell proliferation is achieved.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering and genetic engineering, and relates to the application of miRNA as a target of genetic remodeling in the remodeling and improvement of mammalian host cells. Background technique [0002] The importance of large-scale culture of mammalian cells to produce recombinant protein drugs has become increasingly prominent. In order to meet the research and development of new products and the huge production needs, various strategies of genetic engineering are carried out on Chinese hamster ovary cells (CHO) and other production host cells to overcome many shortcomings that are not suitable for industrial production. It is the basis for the production of recombinant protein drugs by mammalian host cells, and the key is to find targets that can be used for cell engineering. [0003] Using mammalian cells as hosts to produce protein drugs has advantages that host cells such as bacteria and yeast d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85
Inventor 张兵兵鲜成玉徐洪记侯汝涛
Owner CHONGQING UNIV
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