CN116426525A - ASO for inhibiting chicken Wnt3a gene expression and application thereof - Google Patents

ASO for inhibiting chicken Wnt3a gene expression and application thereof Download PDF

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CN116426525A
CN116426525A CN202310300197.XA CN202310300197A CN116426525A CN 116426525 A CN116426525 A CN 116426525A CN 202310300197 A CN202310300197 A CN 202310300197A CN 116426525 A CN116426525 A CN 116426525A
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aso
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wnt3a
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CN116426525B (en
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姬改革
束婧婷
巨晓军
章明
单艳菊
屠云洁
刘一帆
邹剑敏
盛中伟
肖芹
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Jiangsu Institute Poultry Sciences
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Abstract

The invention belongs to the field of gene function research, and particularly relates to ASO capable of inhibiting chicken Wnt3A (Wnt family member 3A) gene expression and application thereof. The invention discloses ASO for inhibiting chicken Wnt3a gene expression, and the nucleotide sequence of the ASO is 5'-AGUGCAGUUCAUUAGCUUCU-3'. After the ASO for inhibiting the expression of the chicken Wnt3a gene provided by the invention is transferred into chicken embryo skin tissues cultured in vitro, the expression of the chicken Wnt3a gene can be effectively reduced, and the inhibition efficiency is more than 50%. The ASO sequence of the invention can identify the gene function by specifically knocking down the expression of the chicken Wnt3a gene, can be applied to genetic breeding of high-quality chickens, the color of bird feathers and provide reference data for human disease research, and has great economic value and scientific research value.

Description

ASO for inhibiting chicken Wnt3a gene expression and application thereof
Technical Field
The invention belongs to the field of gene function research, and particularly relates to ASO capable of inhibiting chicken Wnt3A (Wnt family member 3A) gene expression and application thereof.
Background
In recent years, the poultry industry has entered an important revolution with the emphasis of adverse effects of avian influenza events on the poultry industry and the public's emphasis on food safety. Gradual closing of the live chicken market and market of chilled fresh chickens after slaughter have become an irreversible trend. The appearance of chicken carcasses is gradually an important focus of consumers, and the hair follicle density and the diameter are important character characteristics of the appearance of slaughtered chicken carcasses. Consumers often prefer the appearance of dense and small skin hair follicle carcasses. Thus, the size of the skin follicle will be a major concern for breeding workers and enhance the trait of choice.
The Wnt3A (Wnt family member 3A) gene belongs to Wnt gene family and plays an important role in the growth and development of hair follicles. Wnt3a plays an important role in the size of chicken hair follicles, and Wnt3a can increase the thickness of dermis, enlarge the size of spinal cord bundles, reduce bud intervals, influence the type of bird feathers and the color of feathers, and play an important role in regulating and controlling the feather sac morphogenesis of yellow-feather chickens in embryo periods. However, up to now, no mechanism for controlling Wnt3a molecules has been elucidated in chickens.
Antisense oligonucleotide (ASO, antisense oligonucleotide) refers to a chemically modified short-chain nucleic acid of 15-25 nucleotides, functions depending on RNase H, and can specifically knock down RNA in cytoplasm and nucleus, which is an RNA interference technique commonly used for silencing lncRNA. The expression of Wnt3a gene in skin tissue cultured in vitro is interfered by siRNA technology in the early stage, the interference efficiency is extremely low, the functional research on Wnt3a gene is not realized, and the regulation and control mechanism for revealing the growth and development related characters of chicken feather hair follicles is further hindered.
Therefore, a small RNA fragment capable of interfering the expression of Wnt3a genes of chicken embryo skin tissues is urgently needed, a regulatory mechanism capable of helping to reveal the growth and development related characters of chicken feather hair follicles is facilitated, and market competitiveness of poultry products in China is improved.
Disclosure of Invention
The invention aims to solve the problem that in the prior art, no small RNA fragment which is optimal and interferes with the expression of the Wnt3a gene of the chicken embryo skin tissue exists, and provides an ASO fragment of a target chicken Wnt3a gene.
In a first aspect, the invention provides an ASO that inhibits expression of a chicken Wnt3a gene, the ASO having a nucleotide sequence of 5'-AGUGCAGUUCAUUAGCUUCU-3' (SEQ ID No. 3).
In a second aspect, the invention also provides a recombinant expression vector, a transgenic cell line or a recombinant bacterium containing the nucleotide sequence of the ASO for inhibiting the expression of the chicken Wnt3a gene.
In a third aspect, the invention also provides a kit containing the ASO for inhibiting the expression of the chicken Wnt3a gene.
Further, the kit also comprises a primer group for detecting the expression of the chicken Wnt3a gene.
In a fourth aspect, the invention also provides the ASO, the recombinant expression vector, the transgenic cell line or the recombinant bacterium, and the kit for identifying the functions of the chicken Wnt3a genes.
Compared with the prior art, the ASO sequence capable of targeting the chicken Wnt3a gene is provided, and can effectively reduce the expression of the chicken Wnt3a gene after being transferred into chicken embryo skin tissues cultured in vitro, the inhibition efficiency reaches more than 50%, and the knocking-down efficiency is high. The ASO sequence of the invention can identify the gene function by specifically knocking down the expression of the chicken Wnt3a gene, can be applied to genetic breeding of high-quality chickens, the color of bird feathers and provide reference data for human disease research, and has great economic value and scientific research value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.
Fig. 1 is an efficiency of Wnt3a gene expression in 3 different ASO-interfered chicken skin tissues cultured in vitro, wherein, the difference is significant (P < 0.05) in the ASO-interfered group compared to NC control group; * Indicating that the difference was very significant (P < 0.01) in ASO-interfered groups compared to NC control groups;
FIG. 2Western blot shows the efficiency of ASO interference in expression of Wnt3a gene protein in chicken skin tissue cultured in vitro, wherein, the ASO interference group is significantly different (P < 0.05) compared with NC control group;
FIG. 3 is a graph showing the effect of real-time fluorescent quantitative PCR detection of ASO interference on expression of CT nnb1mRNA of feather follicle growth marker gene after in vitro cultured chicken skin tissue Wnt3a expression knockdown, wherein ASO interference group is significantly different (P < 0.05) compared with NC control group;
FIG. 4 is a Western blot analysis of the effect of ASO interference on the expression of Ctnnb1 protein of the feather follicle growth marker gene after knockdown of Wnt3a gene expression in chicken skin tissue cultured in vitro, wherein, the ASO interference group shows significant differences (P < 0.05) compared with NC control group;
FIG. 5 is a graph showing the effect of ASO interference on the growth and development of feather follicles after knockdown of Wnt3a gene expression in chicken skin tissue cultured in vitro, as determined by HE staining and slicing.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The following examples were carried out by conventional methods, and the reagents used were all commercially available ones unless otherwise specified.
Example 1 screening of optimal sequence for targeting ASO of chicken Wnt3a Gene
ASO design
According to NCBI online database, obtain chicken Wnt3a gene mRNA sequence information (accession number: NM_ 001171601.3), design 3 ASO sequences of target chicken Wnt3a gene, synthesize by Guangzhou Ruibo Biotechnology Co., ltd, control NC is provided by Guangzhou Ruibo Biotechnology Co., ltd. The sequences of the 3 ASOs designed are shown in Table 1.
Table 1 3 ASO sequences
ASO Sequence (5 '-3') Sequence numbering
Wnt3a aso-1 CUCGAAAUUGGUGUUGGCAC SEQ ID NO.1
Wnt3a aso-2 UGAUAGAGGUCCUUCCAGCU SEQ ID NO.2
Wnt3a aso-3 AGUGCAGUUCAUUAGCUUCU SEQ ID NO.3
Table 2 3 ASO targeting sequences
ASO Targeting sequence (5 '-3') Sequence numbering
Wnt3a aso-1 GTGCCAACACCAATTTCGAG SEQ ID NO.4
Wnt3a aso-2 AGCTGGAAGGACCTCTATCA SEQ ID NO.5
Wnt3a aso-3 AGAAGCTAATGAACTGCACT SEQ ID NO.6
ASO transfection in vitro culture of chick embryo skin tissue
The separation and culture of skin tissue from chick embryo tissue maintains many important biological characteristics and functions of skin in vivo, so that the in vitro culture of skin tissue is widely applied to basic research of molecular, cell biology and biomedicine and has non-replaceable effect in the field of biomedicine. However, the transfection efficiency of exogenous genes into the skin tissue of chick embryos is low relative to cells. In this example, the back skin of an 8-embryo chick was cut into skin pieces of equal size from the back skin of an 8-embryo chick, inoculated in a 24-well plate, and treated with CO 2 Culturing in an incubator for 6 hours, and carrying out transfection by referring to Lipofectamine3000 transfection reagent instructions, wherein the operation steps are as follows:
(1) mu.L of Opti-MEM TM Mixing the culture medium and 15 mu L Lipofectamine3000 reagent;
(2) mu.L of Opti-MEM was taken separately TM The culture medium and 5 mug of ASO reagent are fully and evenly mixed;
(3) Mixing the diluted Lipofectamine3000 reagent and ASO reagent uniformly according to the ratio of 1:1, and incubating for 10-15 minutes at room temperature.
(4) Adding the mixed solution into a 24-hole culture plate according to the amount of 50 mu L of each hole; 3 ASO groups, each with 3 replicate wells.
The group transfected with NC control was designated NC group; the group transfected with ASO-1 fragment, designated ASO-1 group; the group transfected with ASO-2 fragments, designated ASO-2 group; the group transfected with ASO-3 fragment was designated ASO-3 group. After ASO 1 day of transfection, the cultured skin tissues are collected, the interference efficiency of ASO on Wnt3a mRNA expression is detected by adopting a qRT-PCR method, and proper interference fragments are screened. After ASO interference fragments are transfected for 3 days, cultured skin tissues are collected, and the influence of the screened ASO fragments on Wnt3a gene protein expression is detected by Western blot.
The primer pair nucleic acid sequence for detecting the expression of the chicken Wnt3a gene is as follows:
an upstream primer: 5'-ACTGAGAAGGACCTGGTTT-3' (SEQ ID NO. 7)
A downstream primer: 5'-ATGGGAAGTGACGTTACAGA-3' (SEQ ID NO. 8)
The primer pair nucleic acid sequence for detecting the expression of the chicken internal reference ACTB gene is as follows:
an upstream primer: 5'-TGCTGTGTTCCCATCTATCG-3' (SEQ ID NO. 9)
A downstream primer: 5'-TTGGTGACAATACCGTGTTCA-3' (SEQ ID NO. 10)
As shown in FIG. 1, compared with the inhibition efficiency of 3 ASO fragments on Wnt3a gene expression in chicken embryo skin tissue cultured in vitro, the ASO-3 interference efficiency is best, and compared with NC control group, the inhibition efficiency of ASO-3 reaches more than 50%, and the expression of Wnt3a gene mRNA in chicken embryo skin tissue cultured in vitro is extremely obviously inhibited (P < 0.01).
After 72h of transfection, each well was washed 2 times with PBS, RIPA cell lysate (Cell Signaling Technology) was selected to extract total cell proteins, and after BCA assay was performed to determine the concentration of the extracted proteins, western blot experiments were performed using Wnt3a (abcam) and beta-action (abcam) as primary antibodies, and goat anti-mouse IgG (Invitrogen) as secondary antibodies, respectively. As shown in fig. 2, the inhibition efficiency of ASO-3 reached 50% or more, compared with NC control group, and expression of Wnt3a gene protein in chicken skin tissue cultured in vitro was significantly inhibited (P < 0.05).
Subsequent experiments selected ASO-3 to study chicken Wnt3a gene function.
Example 2 Effect of Wnt3a Gene expression on feather follicle growth marker Gene Ctnnb1 Gene expression following knockdown
The back skin of the 8-embryo-age chick embryo was cut into skin pieces of equal size, inoculated into 24-well plates, cultured in a CO2 incubator for 6 hours, and ASO-3 targeting the chick Wnt3a gene and negative control NC were transfected into the chick embryo skin tissue cultured in vitro by means of the transfection reagent Lipofectamine3000, respectively, in accordance with the procedure of example 1 described above, with 3 replicate wells per group. After ASO-3 1 days of transfection, cultured skin tissues were collected and the effect of ASO on the expression of the feather development marker gene Ctnnb1mRNA was detected by qRT-PCR. The primer pair nucleic acid sequence for detecting the chicken Ctnnb1 gene expression is as follows:
an upstream primer: 5'-ACGAGGAAAGAGCGACGGAT-3' (SEQ ID NO. 11)
A downstream primer: 5'-CCTTTCAGAGACTGTGGCACG-3' (SEQ ID NO. 12)
After 3 days of ASO-3 interference fragment transfection, cultured skin tissues are collected, and the influence of the screened ASO fragments on the expression of Ctnnb1 gene protein is detected by Western blot. The specific method is the same as in example 1, and after the BCA method is used for measuring the concentration of the extracted protein, ctnnb1 (proteontech) and beta-action (abcam) are respectively used as primary antibodies, and sheep anti-rabbit and sheep anti-mouse IgG (Invitrogen) are used as secondary antibodies for Western blot experiments.
After the Wnt3a gene expression was inhibited, the results of the change in expression of Ctnnb1mRNA and protein of the feather development marker gene are shown in fig. 3-4, and compared with NC control group, both Ctnnb1mRNA and protein expression were significantly reduced in ASO-3 interference group (P < 0.05).
Example 3 effect of Wnt3a Gene knockdown on feather growth
Back skin of 8-embryo chick embryos was cut into skin pieces of equal size, inoculated into 24-well plates, cultured in a CO2 incubator for 6 hours, and ASO-3 targeting the chicken Wnt3a gene and negative control NC were transfected into in vitro cultured chick embryo skin tissues with the aid of transfection reagent Lipofectamine3000, respectively, in accordance with the procedure described in example 1 above, with 3 replicate wells per group. The skin tissues before and after ASO-3 2 days of transfection were collected, the cultured skin tissues were fixed with 10% formaldehyde solution, dehydrated and paraffin-embedded after 24 hours, and then sectioned and HE stained. The NC control group before transfection was designated NC0 group, and the ASO-3 fragment transfected group was designated ASO0 group; the NC control group after 2 days of transfection was designated NC2 group, and the group transfected with ASO-3 fragment was designated ASO2 group.
The results are shown in FIG. 5, where the NC control and ASO-interfered groups had feather follicle germ projections on the skin surface prior to transfection. After 2 days of transfection, NC group chick embryo skin feather follicles grow normally, a plurality of obvious feather buds can be seen to eject out of the epidermis and extend out of the skin; the ASO interference group chicken embryo feather hair follicle primordium has slow growth speed, no obvious new feather hair follicle appears, and the grown feather bud structure is unclear, which indicates that after Wnt3a gene expression is inhibited, the chicken embryo feather hair follicle primordium growth is inhibited.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.

Claims (5)

1. An ASO for inhibiting expression of a chicken Wnt3a gene, wherein the ASO has a nucleotide sequence of 5'-AGUGCAGUUCAUUAGCUUCU-3' (SEQ ID No. 3).
2.A recombinant expression vector, transgenic cell line or recombinant bacterium comprising the nucleotide sequence of ASO of claim 1 that inhibits the expression of a chicken Wnt3a gene.
3. A kit comprising the ASO of claim 1 that inhibits the expression of a chicken Wnt3a gene.
4. The kit of claim 3, further comprising a primer set for detecting the expression of the chicken Wnt3a gene.
5. Use of the ASO of claim 1, the recombinant expression vector, the transgenic cell line or the recombinant bacterium of claim 2, the kit of claim 3, for identifying the function of the Wnt3a gene in chickens.
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