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RNAi interference fragment, interference carrier, preparation method and application thereof

A technology of interference carrier and fragment, which is applied in the field of preparation of RNAi interference carrier, can solve the problems of high deformity rate of offspring, intolerance to freezing, low success rate of gender identification of inferior embryos, etc., and achieves the effect of convenient operation and low cost

Active Publication Date: 2017-06-16
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, these methods have some shortcomings, which limit the practical application
For example, the separation speed of X and Y sperm is too slow, the semen is expensive and not resistant to freezing, resulting in a higher rate of deformity in offspring
When performing embryo gender identification, some cells are cut from the embryo, and then frozen and thawed will cause certain damage to the embryo, which will reduce the success rate of embryo transfer. Commercialization of animal embryos
In the external environment that controls the fertilization of animals, since there are many factors affecting the external environment of animal sex determination mechanism, regulating a single factor cannot effectively affect sex, and it is difficult to achieve the optimal state of multiple factors in practice by regulating multiple factors at the same time, and The effect of regulating these factors is also related to the physique of the animal, so the effect of controlling sex is not obvious

Method used

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  • RNAi interference fragment, interference carrier, preparation method and application thereof
  • RNAi interference fragment, interference carrier, preparation method and application thereof
  • RNAi interference fragment, interference carrier, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Design and synthesize RNAi interference fragments for interfering with Zfx gene in animal testicular 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 Zfx gene in pig testicular germ cells as an example. details as follows:

[0032] According to the mRNA sequence of the pig Zfx gene (GenBank: KF803247), two siRNA fragments were designed and screened, and then according to the requirements of the pLentiLox3.7 vector and its promoter, restriction sites were added at both ends of the two siRNA fragment sequences , loop and termination sequence, the final form is thymine (T) + sense strand 19nt target sequence + stem-loop structure (TTCAAGAGA) + target sequence reverse complement sequence + RNAPolyIII polymerase transcription termination site (TTTTTT) + XhoI digestion site (GAGCT), the oligonucleotide sequences for the synthesis of two pairs of shRNAi. The two pa...

Embodiment 2

[0037] This example mainly prepares an RNAi interference vector for interfering with Zfx gene in porcine testicular germ cells.

[0038]The RNAi interference vector in this example includes RNAi interference fragments, and the RNAi interference fragments are shRNA1 and shRNA2 synthesized in Example 1. The RNAi interference vector also includes an expression vector, which is connected to the RNAi fragment, and the expression vector can be a viral vector or a non-viral vector, preferably a pLentiLox3.7 vector. The specific preparation method of the RNAi interference carrier is as follows:

[0039] Digestion of pLentiLox3.7 vector.

[0040] The pLentiLox3.7 vector in this step, such as figure 1 shown. The digestion system includes 5 μL of pLentiLox.3.7 vector (500 μg / μL), 5 μL of 10×K buffer, 1.5 μL of XhoI and HpaI restriction endonucleases, and 37 μL of RNase-free water (RNase-free water). After the above enzyme digestion system was prepared, it was placed in a constant tem...

Embodiment 3

[0057] The RNAi interference vector pLentiLox.3.7-shRNA1 prepared in Example 2 was used to carry out the Zfx gene interference test in vitro. Specific steps are as follows:

[0058] The testes of piglets aged 27-32 days were collected, and Sertoli cells and germ cells were separated by two-step enzyme digestion method. Sperm cells were incubated at 32°C, 5% CO 2 , and cultured under the condition of 95% humidity for 24 hours, the constructed interference vector pLentiLox.3.7-shRNA1 was transfected into the germ cells with calcium ion solution as the transfection reagent, and each group was repeated three times. The total RNA of germ cells was extracted 48 hours after transfection, and the mRNA expression level of Zfx gene was detected by real-time fluorescence quantitative PCR (qRT-PCR). Preliminarily determine the interference effect of the interference fragment, and then proceed to the next experiment. Primers required for qRT-PCR as shown in Table 2, figure 2 The mRNA ...

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Abstract

The invention provides an RNAi (ribonucleic acid interference) interference segment and RNAi interference vector for interfering with Zfx gene in animal testicle spermatogenic cells. The RNAi interference vector has the RNAi interference segment; and the RNAi interference segment comprises the following sequence: TGAGG CAGAT GTATC TGAAA TTCAA GAGATTTCAG ATACA TCTGC CTCTT TTTTC or TGCAG AGAAG GCCATTGAAT TTCAA GAGAA TTCAA TGGCC TTCTC TGCTT TTTTC. The RNAi interference segment and RNAi interference vector provided by the invention can interfere with the Zfx gene in animal testicle spermatogenic cells, so that the Zfx gene expression is lowered to indirectly influence the fertilization capacity of the X sperm, thereby achieving the goal of controlling the gender of the pig offsprings.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RNAi interference fragment for controlling animal sex, an RNAi interference carrier, and a preparation method and application of the RNAi interference carrier. Background technique [0002] The purpose of animal sex control is to artificially control the sex of animals before they are born. It is of great significance to realize the sex control of animals, which is mainly reflected in: (1) It can timely eliminate or terminate animals that do not need sex, and improve the production efficiency of animal husbandry (the value of female offspring of dairy cows, dairy goats, and laying hens is high, and the value of males is low; Beef cattle, meat pigs, and broiler chickens grow faster); (2) in the production of transgenic animals, increase the utilization value of animals of the required sex (female animals have high utilization value, and male animals have low utilization value); (3)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/66A61K48/00A61P15/00
Inventor 贾斌宁孟影程波
Owner SHIHEZI UNIVERSITY
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