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RNAi fragment, RNAi vector, preparation method and application of RNAi vector

A technology that interferes with vectors and fragments. It is applied in DNA/RNA fragments, recombinant DNA technology, and the introduction of foreign genetic material using vectors. It can solve the problems of expensive equipment, slow separation speed, and poor vitality, and achieve low-cost effects.

Inactive Publication Date: 2016-01-20
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, according to the principle that there is a difference in the DNA content of mammalian X and Y sperm, using a flow cell retrieval instrument to separate X and Y sperm has the best separation effect, and the purity can reach more than 90%. However, due to expensive equipment and slow separation speed, it is used for Due to the low number of fertilized sperm and poor motility, it is far from meeting the large demand for sexually controlled semen in production.

Method used

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  • RNAi fragment, RNAi vector, preparation method and application of RNAi vector
  • RNAi fragment, RNAi vector, preparation method and application of RNAi vector
  • RNAi fragment, RNAi vector, preparation method and application of RNAi vector

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Embodiment 1

[0034] Design and synthesis of RNAi interference fragments for interference with Zfy gene in animal testis germ cells. In the following example, the technical solution provided by the present invention will be described by taking the RNAi interference fragment of the Zfy gene in the spermatogenic cells of sheep testis as an example. details as follows:

[0035]According to the mRNA sequence of sheep Zfy gene, 8 siRNA fragment templates were designed and two siRNA fragments were screened out, of which Table 1 is the ZfycDNA template used for siRNA;

[0036] Table 1 ZfycDNA template for siRNA

[0037]

[0038]

[0039] Then, according to the requirements of the pLentiLox3.7 vector and its promoter, two pairs of shRNAi oligonucleotide sequences were synthesized. The two pairs of shRNAi interference fragments are shRNAA and shRNAG respectively, as table 2 shown.

[0040] The Zfy gene sequence used was from the NCBI gene bank, and 8 siRNA fragments were screened out ...

Embodiment 2

[0044] The screening method of two pairs of shRNAi interference fragments described in embodiment 1 is specifically as follows:

[0045] 1. Construct the RNAi interference vector of Zfy gene:

[0046] The Zfy gene sequence used was from the NCBI gene bank, and 8 siRNA fragments were screened out according to the RNAi design principles and the comparison analysis results on NCBI, as table 1 Shown is the ZfycDNA template used for siRNA. Add XholI and HpaI restriction sites to the 5' and 3' of each siRNA fragment, respectively, and then send the siRNA fragment to be synthesized by a biological company. The positive and antisense strands were combined into double strands by annealing to the plentilox3.7 (Addgene, USA) lentiviral packaging system vector, and then transformed into E.coliDH5α competent cells (Tiangen, China) for amplification, and the plasmid was extracted- Store at 20°C.

[0047] 2. In vitro interference test of Zfy gene:

[0048] Select 2 testes of 1-year-old ...

Embodiment 3

[0064] An RNAi interference carrier, comprising a RNAi interference fragment whose sequence is shown in SEQ ID NO.1, and its preparation method comprises the following steps:

[0065] (1) Digest the pLentiLox3.7 vector;

[0066] (2) Ligate the RNAi interference fragment with the pLentiLox3.7 carrier after digestion to obtain a ligation product;

[0067] Wherein, the RNAi interference fragment sequence is SEQ ID NO.1;

[0068] (3) Transform the ligation product into competent cells, screen with the pLentiLox3.7 vector, restriction site and hairpin structure, and perform sequencing identification on the obtained bacterial liquid, and compare the sequencing results with the RNAi Amplify the bacteria solution with the same sequence of the interference fragment;

[0069] The enzyme cutting sites are HpaI and XholI; the hairpin structure sequence is SEQ ID NO.3;

[0070] (4) Extracting the RNAi interference carrier from the amplified bacterial liquid.

[0071] The vector constru...

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Abstract

The invention provides an RNAi fragment, an RNAi vector, a preparation method of the RNAi vector and application of the RNAi vector and relates to the technical field of biology. The RNAi fragment is used for interfering Zfy genes on sheep chromosomes Y and characterized in that the sequence of the RNAi fragment is shown in SEQ ID NO.1 or SEQ ID NO.2; protein, in charge of encoding, of the Zfy genes is used as a transcription factor and is related to occurrence of sperms. The RNAi fragment can block or hurt or damage normal development and functions of sperms Y by silencing the Zfy genes in the germ cell sperms Y of male sheep.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RNAi interference fragment, an RNAi interference carrier, and a preparation method and application of the RNAi interference carrier. Background technique [0002] Under natural conditions, the sex ratio of almost all mammals is close to 1:1, thus achieving uninterrupted and stable reproduction of offspring and achieving ecological balance under natural conditions. For the actual production of livestock, the ideal sex ratio is of great significance. Production enterprises using lactation and egg production traits hope to obtain more female individuals, while those with meat production and hair production traits have more obvious advantages in male individuals. Humans have always been interested in controlling the sex of animal offspring. At present, gender control is mainly carried out from two aspects: pre-fertilization and post-fertilization. The former uses sperm separation an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/66A61K48/00A61P15/00
Inventor 贾斌张永生王绪海申红
Owner SHIHEZI UNIVERSITY
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