KR-20260067480-A - Vector for identifying mesodermal cell fate and technique for improving efficiency of cultured meat production using said vector

Abstract

The present invention relates to a method for detecting differentiation from stem cells into sarcoaxial mesenchymal cells using a vector comprising a promoter and a reporter gene of MSGN1, and more specifically, to a vector comprising a promoter and a reporter gene comprising at least one of the transcription factor binding sites represented by SEQ ID NOs 1 to 8, a method for detecting differentiation from stem cells into sarcoaxial mesenchymal cells using the same, a substance that promotes differentiation from stem cells into sarcoaxial mesenchymal cells, a screening method for a differentiation marker from stem cells into sarcoaxial mesenchymal cells, a method for isolating sarcoaxial mesenchymal cells, and a method for producing cultured meat using the same. When using a vector comprising a promoter and a reporter gene that includes one or more of the transcription factor binding sites represented by SEQ ID NOs 1 to 8 of the present invention, the degree of differentiation from stem cells into sarcoaxial mesenchymal cells, the state and timing of differentiation induction, etc., can be detected quickly and accurately. By using the vector, differentiation markers and differentiation-promoting substances for sarcoaxial mesenchymal cells can be screened, sarcoaxial mesenchymal cells can be effectively isolated, and ultimately, the production efficiency of cultured meat can be maximized.

Inventors

• 최광환
• 이동경
• 이창규
• 정진솔
• 안예림

Assignees

• 주식회사 스페이스에프
• 서울대학교산학협력단

Dates

Publication Date

20260513

Application Date

20241105

Claims (13)

1. A vector comprising a promoter and a reporter gene, each comprising one or more transcription factor binding sites represented by SEQ ID NOs 1 to 8.
2. A vector according to claim 1, characterized in that the promoter includes sequence number 9.
3. The method of claim 1, wherein the reporter gene is mEGFP, EGFP, GFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Sapphire, EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean 433, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1 (Teal), EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), A vector characterized by being one or more selected from the group consisting of DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143.
4. Stem cells transformed by a vector according to paragraphs 1 to 3.
5. In paragraph 4, a stem cell characterized by being of animal origin.
6. A stem cell according to claim 5, characterized in that the animal is one or more selected from the group comprising pig (porcine), bovine (bovine), horse (equine), pig, sheep (ovine), elk, bison (bison), rabbit (Leporidae), deer (Cervidae), dog (Caninae), chicken (Gallus gallus domesticus), duck (Anas), and turkey (Meleagris).
7. A stem cell characterized as being an embryonic stem cell in paragraph 4.
8. A composition for detecting differentiation from stem cells into sarcoaxial mesenchymal cells, comprising a vector according to claims 1 to 3.
9. A method for detecting differentiation from stem cells into sarcoaxial mesenchymal cells comprising the following steps: A step of transforming stem cells with a vector according to claims 1 to 3; and A step of detecting differentiation into sarcoaxial mesenchymal cells by measuring the expression of the above reporter gene.
10. Screening method for substances that promote differentiation from stem cells into sarcoaxial mesenchymal cells, comprising the following steps: A step of transforming a vector according to paragraphs 1 to 3 into a stem cell; A step of culturing stem cells transformed with the above vector in the presence of a candidate substance; and A step of measuring reporter gene expression of the cultured stem cells; A step of determining that the candidate substance is a substance that promotes differentiation from stem cells into sarcoaxial mesenchymal cells when the expression of the reporter gene is increased compared to the case where there is no candidate substance.
11. Screening method for differentiation markers from stem cells to sarcoaxial mesenchymal cells comprising the following steps: A step of transforming each stem cell with a vector according to paragraphs 1 to 3; A step of comparing gene expression between a cell expressing the reporter gene and a cell not expressing the reporter gene; and A step of selecting a gene specifically expressed in the cell in which the above reporter gene is expressed as a differentiation marker.
12. A step of transforming each stem cell with a vector according to claims 1 to 3; and A method for isolating sarcoplastic cells comprising the step of isolating cells expressing the above reporter gene.
13. A method for producing cultured meat comprising the step of culturing cells separated by the method according to paragraph 12.

Description

Vector for identifying mesodermal cell fate and technique for improving efficiency of cultured meat production using said vector The present invention relates to a method for detecting differentiation from stem cells into sarcoaxial mesenchymal cells using a vector comprising a promoter and a reporter gene of MSGN1, and more specifically, to a vector comprising a promoter and a reporter gene comprising at least one of the transcription factor binding sites represented by SEQ ID NOs 1 to 8, a method for detecting differentiation from stem cells into sarcoaxial mesenchymal cells using the same, a substance that promotes differentiation from stem cells into sarcoaxial mesenchymal cells, a screening method for a differentiation marker from stem cells into sarcoaxial mesenchymal cells, a method for isolating sarcoaxial mesenchymal cells, and a method for producing cultured meat using the same. Pluripotent stem cells (PSCs), including embryonic stem cells (ES cells) and induced pluripotent stem cells (iPSCs), can differentiate into skeletal muscle through the regulation of signaling pathways that mimic in vivo muscle development, which is emerging as a breakthrough for sustainability, particularly in cultured meat production. However, heterogeneity resulting from the induction into non-myogenic cell types during the differentiation process leads to low differentiation efficiency. To improve meat production yield, a reporter system based on the expression of mesogenin1 (MSGN1) in the paraxial mesoderm, which is an intermediate state between pluripotent stem cells and myogenic lineage, can be applied. As a major marker of the sarcoaxial mesoderm, particularly the posterior presomitic mesoderm (PSM), MSGN1 is involved in early muscle development. During embryonic development, the expression of Wnt3a, T, and Tbx6 leads to MSGN1 expression for lineage specification of the PSM in the posterior region. Subsequently, MSGN1, a transcriptional regulator, induces the expression of genes involved in Notch, FGF, and WNT signaling oscillations for somitogenesis. This MSGN1-mediated regulatory network of the sarcoaxial mesoderm is a prerequisite for myogenesis in somites. Considering the functional locus of MSGN1 as a transcription factor, the isolation of the sarcoaxial mesoderm can be achieved through a fluorescence reporter system based on the MSGN1 upstream region. While the function of MSGN1 is conserved across various species, the sequences of the upstream regions, including the promoter, are species-specific. Indeed, although it has been demonstrated that most binding sites for WNT signaling and the Tbx6 transcription factor are located within 1.2 kb of the MSGN1 promoter, the nucleotide sequences of these regions themselves exhibit low homology between species. This suggests that a reporter system composed of porcine species-specific MSGN1 promoter sequences is essential for the proper functioning of the system in pigs. Unlike mice and humans, despite their importance in developmental research and the agricultural industry, there are no studies on MSGN1 reporter systems for livestock, including pigs, or on their application to PSC differentiation-based cultured meat production. Since reporter systems can identify the fate of sarcoaxial mesoderm cells, the present invention ultimately aims to improve the efficiency of cultured meat production by using a porcine MSGN1 promoter-based reporter in the following processes: (1) monitoring of the differentiation process, (2) screening of differentiation-inducing candidate substances, (3) discovery of sarcoaxial mesoderm-specific lineage markers, and (4) cell enrichment through fluorescence signal-based cell sorting. The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs. Figure 1 shows a vector map of the vector of the present invention. A vector carrying a fluorescent reporter gene under the MSGN1 promoter sequence was constructed and used in a mesenchymal cell fate identification system. Figure 2 illustrates the results of the genomic integration validation of the reporter system. Figure 2(A) shows the PCR results using genomic DNA from pig ES cells transfected with the reporter system (pMSGN1 R-ES): the pMSGN1 vector was the positive control, and untransfected water and pig ES cells (WT) were the negative controls. Arrows indicate the pMSGN1 vector (228 bp) and the loading control ACTB (126 bp). WT: wild-type, pMSGN1 R-ES: MSGN1 promoter-based reporter-transfected pig ES cells. Figure 2(B) shows the sequencing results of the PCR products amplified using inserted vector-specific primers: regions that match the target sequence 100% (sequencing region 1 with the forward primer and region 2 with the reverse primer). Figure 3 shows the results of characterizat