CN109097400B

CN109097400B - Method for activating expression of endogenous Pdx1 gene based on chromatin remodeling

Info

Publication number: CN109097400B
Application number: CN201810947480.0A
Authority: CN
Inventors: 王启伟; 叶华虎
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2018-08-20
Filing date: 2018-08-20
Publication date: 2022-01-04
Anticipated expiration: 2038-08-20
Also published as: CN109097400A

Abstract

The invention discloses a method for activating expression of an endogenous Pdx1 gene based on chromatin remodeling. The invention provides a method for activating expression of an endogenous Pdx1 gene in a target cell, which is a method for activating expression of the endogenous Pdx1 gene in the target cell based on a CRISPR-on SAM system. The SAM system comprises sgRNA which targets the position from-400 bp to +1bp upstream of the Pdx1 gene transcription starting point, and the target sequence of the sgRNA is SEQ ID No.3 and/or SEQ ID No. 4. The invention applies CRISPR-on technology and can efficiently activate the expression of Pdx1 of 293T cells through chromatin remodeling. The research plays an important role in the research of directionally differentiating the PSCs into beta cells and the embryonic development of pancreas by gene induction.

Description

Method for activating expression of endogenous Pdx1 gene based on chromatin remodeling

Technical Field

The invention relates to the technical field of biology, in particular to a method for activating expression of an endogenous Pdx1 gene based on chromatin remodeling.

Background

The incidence of diabetes is increasing dramatically year by year. The WHO report in 2017 shows that the incidence rate of diabetes around the world is about 8.5%, and about 4.22 hundred million diabetics exist. Type 1diabetes is caused by the attack of the islet beta cells by an autoimmune response. Clinical symptoms are indicated when the number of islet beta cells remaining is less than 10-20% of the total number of islet cells [ Gillespie, K.M.Type 1diabetes: pathogenesis and preservation. CMAJ 175,165-170, doi:10.1503/cmaj.060244(2006) ]. Type 2diabetes is characterized by a decrease in cell proliferation and increased apoptosis that leads to abnormal beta cell function and progressive cell number reduction [ Joost, H.G. Patholonesis, risk assessment and prevention of type 2diabetes mellitus. Obes effects 1, 128-. Currently, islet beta cell transplantation is considered to be one of the most effective approaches for treating diabetes. However, the application of large-scale islet beta cell transplantation is limited by the shortage of cell sources and the lifetime use of immunosuppressive agents [ Atkinson, M.A. & Eisenbarth, G.S. type 1diabetes, new perspectives on disease pathogenesis and treatment. Lancet 358,221-229, doi:10.1016/S0140-6736(01)05415-0(2001) ]. Pluripotent Stem Cells (PSCs) include Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs), and have self-renewal and multipotent differentiation potential. Thus, PSCs are ideal cell sources for cellular therapy of diabetes. In recent years, the research on the directed differentiation of PSCs into islet beta cells and non-islet beta cells has been advanced [ Miyazaki, S., Yamato, E. & Miyazaki, J.regulated expression of pdx-1 proteins in vitro differentiation of insulin-producing cells from diabetes cells 53,1030 + 1037(2004) ]. The current methods for inducing cell differentiation into insulin-secreting cells mainly include two categories: firstly, a specific transcription factor method for regulating islet beta cell generation is adopted; the other method is a combination method of growth factors and small molecular compounds. The gene inducing method mainly adopts key transcription factor in the islet cell generating process to induce cell differentiation and transdifferentiation. Miyazaki et al constructed an ES cell line for inducing expression of Pdx1 by using a Tet off system, and showed that Pdx1 can improve the efficiency of differentiation of ESCs into beta cells by using growth factors and small molecule compounds, and enhance the expression of genes related to differentiated cells, such as insulin 2, somastatin, Kir6.2, glucokinase, neurogenin3, p48, Pax6, PC2 and HNF6[ Miyazaki, S., Yamato, E. & Miyazaki, J.regulated expression of Pdx-1 proteins in vitro differentiation of insulin-producing cells from embryo fibrous cells. diabetes 53,1030- & 1037(2004) ]. Kubo et al showed that overexpression of Pdx1and Ngn3 significantly upregulated endoderm cells expressing insulin and islet-associated genes [ Kubo, A.et al. Pdx1and Ngn3 overexpression enhancement of vascular endothelial cell-derived end expression. PLoS One 6, e24058, doi: 10.1371/joural. point.0024058 (2011) ]. The adenovirus expressing Pdx1 can infect human skin keratinocytes, can rapidly activate pancreas to generate related specific transcription factors, and can induce and differentiate the cells into insulin secreting cells. These transdifferentiated cells have the function of synthesizing and secreting insulin under glucose stimulation [ Mauda-Havakuk, M.et al.Ectopic PDX-1expression direct expressions human insulin genes human insulin secretion along with recombinant insulin-producing cell failure. PLoS One 6, e26298, doi: 10.1371/journel.p.0026298 (2011) ]. Zhou et al found that Pdx1, Ngn3, and MafA converted pancreatic exocrine cells to islet beta cells in mice. These transformed cells, which are very similar to endogenous islet beta cells, express genes related to islet beta cell function and are capable of secreting insulin and lowering blood glucose in mice [ Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D.A. in vivo reprogramming of adult pancreatic islet extracellular cells to beta-cells. Nature 455,627-632, doi:10.1038/nature07314(2008) ]. Akinci et al similarly observed the transdifferentiation of the rat pancreatic exocrine cell line AR42j-B13 by Pdx1, Ngn3and MafA in vitro. Reducing blood glucose levels in diabetic mice despite the expression of insulin by the cells obtained by transdifferentiation; however, Pdx1, Ngn3and MafA endogenous to the cells are not activated, and some genes related to the functions of islet beta cells are not expressed. Thus, the transdifferentiation of these cells is incomplete [ Akinci, E.A., Banga, A., Greder, L.V., Dutton, J.R. & Slack, J.M.Reprogramming of systematic exosporine cells towards a beta (beta) cell charater using Pdx1, Ngn3and MafA.biochem J442, 539-. The method for inducing and differentiating the genes has strong specificity, relatively easy operation and lower cost. However, the expression of endogenous related genes is difficult to activate by the current method of exogenous gene overexpression, and thus, differentiated cells do not have mature physiological functions. The cell differentiation of each lineage in embryonic development is precisely controlled by lineage specific transcription factors and finally differentiated into terminally differentiated cells of each germ layer. Meanwhile, the activation of various transcription factors during cell differentiation is controlled by various epigenetic regulatory mechanisms. Epigenetic factors and various transcription factors are coordinated with each other to form a microenvironment suitable for specific lineage differentiation, so that normal differentiation of various cells of an organism can be effectively ensured. The important role of epigenetics in cell function can be substantiated from the process of cell reprogramming. The process of reprogramming fibroblasts to iPSCs, an important factor affecting the reprogramming efficiency, is the epigenetic barrier. The Huanggfu et al studies show that DNA methyltransferase and histone deacetylase inhibitors can significantly improve the efficiency of cell reprogramming. In particular, histone deacetylase inhibitor valproic acid (VPA) can improve the reprogramming efficiency by 100 times [ Huanggfu, D.et al.Induction of complex stem cells by defined factors is great improved by small-molecule-molecular compounds. Nat Biotechnol26,795-797, doi:10.1038/nbt1418(2008) ]. Recently, the Ding group has reset the epigenetic status of Chromatin Oct4 and Sox2 sites by CRISPR (clustered regulated interspersed short palindromic repeats) technique, activating the expression of Endogenous Oct4 and Sox2, successfully Reprogramming fibroblasts to iPSCs [ Liu, P., Chen, M., Liu, Y., Qi, L.S. & Ding, S.CRISPR-Based Chromatin remodelling of the endogenesis Oct4 or Sox2 cells engineering plura. Cell Stem Cell 22,252 gambling 261e254, doi:10.1016/j.stem.2017.12.001(2018) ], which further shows the important role of epigenetic in Cell function.

Pdx 1is a homeodomain transcription factor that plays an important role in early embryonic pancreatic development, endocrine cell formation and late beta cell maturation [ Pan, F.C. & Wright, C.Pancreas oncogenesis: from bud to plexus to gland. Dev Dyn 240,530-565, doi:10.1002/dvdy.22584(2011) ]. The mouse Pdx1 was first expressed at day 8.5 of the embryo (E8.5) [ Miki, R.et al. fat maps of ventral and nasal secretion promoter cells in early tolerance stage mice development.Mech Dev 128,597-609, doi:10.1016/j.mod.2011.12.004(2012) ], and the human Pdx1 was first expressed at 4 weeks of gestation (G4w) [ Jennings, R.E.et al. development of the human pancreas from for expression of endocrine communition.Diabeteses 62,3514-3522, doi: 10.2337/12-1479 (2013) ]. Early embryonic pancreatic development in rodents requires expression of Pdx1, and Pdx1 deficient mice fail to form the pancreas and die within days after birth [ Jonsson, j., Carlsson, l., Edlund, T. & Edlund, h.insulin-promoter-factor 1is required for pancreas reduction in micro 609, doi:10.1038/371606a0(1994) ]. Subsequent development of each pancreatic lineage still requires sustained expression of Pdx 1. In the formation of endocrine cells, the highly expressed Pdx1 plays an important role in the committed differentiation of cells [ Pan, F.C. & Wright, C.Pancreas organogenesis: from bud to plexus to gland. Dev Dyn 240,530-565, doi:10.1002/dvdy.22584(2011) ]. In adults, Pdx 1is expressed only in beta and delta cells of pancreatic islets, and maintains the characteristics and functions of beta cells by regulating genes associated with glucose homeostasis [ Gao, T.et al.Pdx1mainains beta cell identity and function by predicting an alpha cell program, cell Metab 19,259-271, doi:10.1016/j.cmet al.2013.12.002 (2014) ]. In conclusion, Pdx1 plays an important role in embryonic pancreatic development and in vitro induced cell directed differentiation into beta cells. Therefore, the establishment of an in vitro efficient endogenous Pdx1 activation method plays an important role in the research of the directional differentiation of the gene-induced PSCs into beta cells and the embryonic development of pancreas.

CRISPR technology is another powerful genome editing technology following Zinc Finger Nucleases (ZFNs) and TALENs (transcription activator-like effectors) and is used in the field of genetic engineering. The engineered CRISPR/Cas9 consists of sgRNA (tracrRNA: crRNA) and Cas9. Cas9 has two functional domains, HNH and RuvC, with endonuclease activity. Both domains were mutated simultaneously (H840A and D10A mutations), and Cas9 lost endonuclease activity (inactivated Cas9, dCas9) and converted to sgRNA-directed DNA binding protein [ Doudna, J.A. & Charpentier, E.genome edition.the new front of genome engineering with CRISPR-Cas9.science 346,1258096, doi: 10.1126/science.8012596 (2014) ]. dCas9 is fused with a transcription activator, and the fusion is combined with a promoter region of a gene under the guidance of sgRNA, so that the expression of an endogenous gene can be strongly activated, and the defect that the endogenous gene is difficult to activate in gene induction expression is effectively overcome [ Konermann, S.et al genome-scale transcriptional activation by an engineered CRISPR-Cas9 complete. Nature 517, 583. about. 588, doi:10.1038/nature14136(2015) ].

Disclosure of Invention

In order to effectively solve the technical problems, the invention aims to provide a method for activating the expression of an endogenous Pdx1 gene based on chromatin remodeling. The invention applies a SAM (systematic identification activation media) system based on CRISPR-on to establish a high-efficiency endogenous Pdx1 activation method. The SAM system consists of three parts, sgRNA, NLS-dCas9-VP64 and MS2-P65-HSF 1. When the SAM system is expressed in cells, the three components form a transcription activation complex, which, in combination with the specific promoter region targeted by the sgRNA, activates gene expression.

In a first aspect, the invention claims a method of activating expression of an endogenous Pdx1 gene in a target cell.

The method for activating the expression of the endogenous Pdx1 gene in the target cell is a method for activating the expression of the endogenous Pdx1 gene in the target cell based on a CRISPR-on (CRISPR-activated) SAM system.

Further, the SAM system includes sgRNA targeting a position-400 to +1bp upstream of a Transcription Start Site (TSS) of Pdx1 gene, the target sequence of the sgRNA being SEQ ID No.3 (corresponding to sgRNA3 in the present examples) and/or SEQ ID No.4 (corresponding to sgRNA4 in the present examples).

Still further, the method may comprise the steps of: allowing the target cell to express dCAS-VP64 fusion protein, MS2-P65-HSF1 fusion protein, and the sgRNA (dCAS-VP64 fusion protein, MS2-P65-HSF1 fusion protein, and the sgRNA form a SAM complex), thereby activating endogenous Pdx1 gene expression in the target cell.

More specifically, the method may comprise the steps of:

(1) packaging a recombinant lentivirus A capable of expressing the dCAS-VP64 fusion protein; packaging a recombinant lentivirus B capable of expressing the MS2-P65-HSF1 fusion protein; then infecting the target cells with the recombinant lentivirus A and the recombinant lentivirus B together to obtain a positive cell line;

(2) introducing a vector capable of expressing the sgRNA into the positive cell line obtained in the step (1), and further activating the expression of an endogenous Pdx1 gene in the target cell.

In the step (1), when the recombinant lentivirus A is packaged, the adopted target plasmid can be a lenti dCAS-VP64_ Blast vector; when the recombinant lentivirus B is packaged, the adopted target plasmid can be MS2-P65-HSF1_ Hygro vector. When the recombinant lentivirus A and the recombinant lentivirus B are packaged, the adopted helper plasmids can be PMD2.G plasmids and PsPax2 plasmids; the packaging cells used can be 293T cells.

In the step (2), the vector capable of expressing the sgRNA is a vector A and/or a vector B. The vector A is a recombinant vector obtained by inserting a DNA fragment shown in SEQ ID No.3 into a lenti sgRNA (MS2) _ zeo backbone through a BsmB I enzyme cutting site. The vector B is specifically a recombinant vector obtained by inserting a DNA fragment shown in SEQ ID No.4 into a lenti sgRNA (MS2) _ zeo backbone through a BsmB I enzyme cutting site.

In a specific embodiment of the present invention, the vector capable of expressing the sgRNA is the vector a and the vector B, and the mass ratio of the vector a to the vector B is 1:1 (for example, 0.8 μ g of the two vectors are introduced into the target cell in each direction, and the target cell is inoculated on the previous day in an inoculation amount of 3 × 10⁵)。

In a specific embodiment of the present invention, the method comprises the steps of: (1) introducing a lenti dCAS-VP64_ Blast vector, a PMD2.G plasmid and a PsPax2 plasmid (the mass ratio of the lenti dCAS-VP64_ Blast vector to the PsPax2 plasmid can be 1: 0.25: 0.75) into 293T cells, and packaging to obtain the recombinant lentivirus A; introducing an MS2-P65-HSF1_ Hygro vector, a PMD2.G plasmid and a PsPax2 plasmid (the mass ratio of the three can be 1: 0.25: 0.75) into 293T cells, and packaging to obtain the recombinant lentivirus B; and then infecting the target cells by the recombinant lentivirus A and the recombinant lentivirus B together to obtain a positive cell line. (2) Introducing the vector A and the vector B (the mass ratio is 1:1, further, if 0.8 mu g of each of the two vectors is introduced into the positive cell line obtained in the step (1), inoculating the target cells in the previous day in an inoculation amount of 3 x 10⁵) Thereby activating the expression of endogenous Pdx1 gene in the target cell.

In a particular embodiment of the invention, the target cells are in particular 293T cells. Of course, the target cell may also be Pluripotent Stem Cells (PSCs), such as Embryonic Stem Cells (ESCs) or Induced Pluripotent Stem Cells (iPSCs), as desired.

In a second aspect, the invention claims a method for producing a cell in which expression of the endogenous Pdx1 gene is activated.

The method for preparing the cells with activated endogenous Pdx1 gene expression provided by the invention can comprise the following steps: cells in which expression of the endogenous Pdx1 gene is activated are prepared using the method described in the first aspect above.

In a third aspect, the invention claims any one of the following biomaterials:

(I) cells in which expression of the endogenous Pdx1 gene has been activated are prepared by the method described in the second aspect above.

(II) a sgRNA as described in the first aspect hereinbefore.

(III) a vector or a set of vectors;

the vector is the "vector capable of expressing the sgRNA" as described in the first aspect above (the vector a and/or the vector B).

The set of vectors consists of the "vector capable of expressing the sgRNA" described in the first aspect, the lenti dCAS-VP64_ Blast vector, and the MS2-P65-HSF1_ Hygro vector. Of course, pmd2.g plasmids and PsPax2 plasmids may also be included.

In the second and third aspects above, the cell may be a 293T cell, or may be a pluripotent stem cell (e.g. an embryonic stem cell or an induced pluripotent stem cell).

In a fourth aspect, the invention also claims the use of any one of:

(A1) use of a method as hereinbefore described in the first aspect for inducing directed differentiation of pluripotent stem cells into islet beta cells;

(A2) use of a method as hereinbefore described in the first aspect or a cell as hereinbefore described in the second aspect for promoting pancreatic embryo development.

Wherein, the pluripotent stem cells can be embryonic stem cells or induced pluripotent stem cells.

The invention applies CRISPR-on technology and can efficiently activate the expression of Pdx1 of 293T cells through chromatin remodeling. The invention plays an important role in the directional differentiation of the gene-induced PSCs into beta cells and the research on the embryonic development of pancreas.

Drawings

Fig. 1 shows Pdx1 promoter and sgRNA design. TSS is the transcription initiation point. Black arrows indicate relative positions of sgrnas in the promoter region, and numbers indicate the positions of sgrnas with respect to TSS. sgRNA1, sgRNA4 and sgRNA5 target the sense strand of the Pdx1 gene, and sgRNA2 and sgRNA3 target the antisense strand of the Pdx1 gene.

FIG. 2 is Pdx 1expression analysis. A is RT-PCR detection of Pdx1 gene expression. 3 days after transfection of 293T cells with sgrnas, total RNA was extracted and analyzed. B is qPCR detection of Pdx1 gene expression. Changes in Pdx 1expression levels were analyzed 3 days after sgRNA transfection of 293T cells. Gene expression data were normalized to GAPDH transcript levels. Experimental results were confirmed by three independent biological replicates,. p < 0.05; p < 0.01; p < 0.001.

Fig. 3 is sgRNA synergy assay. A is RT-PCR detection of Pdx1 gene expression. 3 days after transfection of 293T cells with sgrnas, total RNA was extracted and analyzed. B is qPCR detection of Pdx1 gene expression. Changes in Pdx 1expression levels were analyzed 3 days after sgRNA transfection of 293T cells. Gene expression data were normalized to GAPDH transcript levels. Experimental results were confirmed by three independent biological replicates,. p < 0.05; p < 0.01; p < 0.001.

FIG. 4 is an immunofluorescence assay. Cells 3 days after sgRNA3+4 transfection were collected, cell flail was prepared, anti-Pdx1(1:100) staining was performed, and INS-1 cells were used as a positive control group. Nuclei were counterstained with DAPI.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 activation of endogenous Pdx1 Gene expression based on chromatin remodeling

Materials and methods

CRISPR vector

From Addge, lenti dCAS-VP64_ Blast (hereinafter referred to as dCAS-VP64), lenti MS2-P65-HSF1_ Hygro (hereinafter referred to as MPH), and lenti sgRNA (MS2) _ zeo backbone were purchased. The Addgene IDs for the three vectors are 61425, 61426, and 61427, respectively.

(II) lentiviral packaging plasmids PMD2.G and PsPax2(Addgene) and 293T cells (Invitrogen), rat insulinoma INS-1 cells (AddexBio).

(III) design of sgRNA specifically bound to promoter and vector construction

1. sgRNA design

5 sgrnas were designed-400 to +1bp upstream of the Pdx1 gene Transcription Start Site (TSS) using CRISPR-ERA (CRISPR-ERA. stanford. edu) online software provided by Lei Qi laboratories (fig. 1). The target sequences of sgrnas are shown in table 1. The sgRNA target sequence oligonucleotide was synthesized from huada gene.

Table 1 sgRNA targeting sequence and position on Pdx1 promoter

sgRNA	Target sequence (5 '-3')	PAM	Chain of the place	Position of distance TSS
					1	GCGGAGCTGTCAAAGCGAGC(SEQ ID No.1)	AGG	+	-22/-3
2	GTTCAGCCGGGGGCCGTGAT(SEQ ID No.2)	TGG	-	-103/-122
					3	GCCTGGCTGGCCGCACTAAG(SEQ ID No.3)	AGG	-	-125/-144
4	GCAGGTGCTCGCGGGTACCT(SEQ ID No.4)	GGG	+	-175/-156
					5	GTTTTCGTGAGCGCCCATTT(SEQ ID No.5)	TGG	+	-266/-247

2. sgRNA vector construction

(1) sgRNA oligonucleotide chain annealing treatment

Two complementary sgRNA oligonucleotide single strands synthesized were treated with ddH₂O was diluted to 100. mu.M. Then, annealing treatment was performed under the following reaction conditions to synthesize double-stranded sgrnas (the sequence of the double-stranded portion was the target sequence in table 1, and both ends were sticky ends conforming to the BsmB I nicks).

Reaction system: sgRNA-Forward (100. mu.M) 1. mu.l; sgRNA-Reverse (100. mu.M) 1. mu.l; 10 XT 4DNA ligase buffer (NEB) 1. mu.l; ddH₂O 7μl。

Reaction conditions are as follows: 30min at 37 ℃; 95 ℃ for 5 min; at 90 ℃ for 1 min; thereafter, gradient annealing was carried out at 5 ℃/min until 4 ℃.

After the reaction is finished, the product is treated with ddH₂Diluted 200 times with O for subsequent support ligation reactions.

(2) Carrier ligation reaction

The lenti sgRNA (MS2) _ zeo backbone vector was cleaved with enzyme, recovered and purified with a gel, and then subjected to ligation reaction.

An enzyme digestion reaction system: lenti sgRNA (MS2) _ zeo backbone vector 2. mu.l; 10 × Buffer 3.1(NEB)5 μ l; BsmB I (NEB) 2. mu.l;ddH₂o41. mu.l. Reaction conditions are as follows: 55 ℃ for 2 hrs.

And (3) connection reaction: 1 μ l of double-stranded sgRNA; the digestion product of lenti sgRNA (MS2) _ zeo backbone vector was 1. mu.l; ddH₂O3 mu l; 2 × solution I (Takara)5 μ l. Reaction conditions are as follows: 22 ℃ for 2 hrs.

(3) Transformation of

And transforming and plating the ligation product, selecting positive clones, and sequencing and identifying.

(IV) dCAS-VP64/MPH cell line construction and screening

1. Lentiviral preparation

The procedure for packaging dCAS-VP64 and MPH lentivirus is briefly described as follows: the day before virus preparation, according to 4-5X 10⁶293T cells were seeded at a density of/10 cm dishes. The day of virus preparation, the gene vector of interest dCAS-VP64 or MPH and lentiviral packaging plasmids pmd2.g and PsPax2 were mixed as follows: 0.25: 0.75 ratio (mass ratio), 293T cells were co-transfected with Lipofectamine2000 (Invitrogen). Culture medium supernatant containing virus particles was collected at 48-72hrs after transfection, and centrifuged at 1500rpm to remove cells and debris suspended in the supernatant. The virus was then concentrated by centrifugation at 20000rpm for 2hrs at 4 ℃. After centrifugation, the supernatant was discarded and the viral particles were resuspended in 4ml of medium for further use. Two recombinant lentiviruses were obtained by this procedure.

2. Construction of 293T cell line expressing dCAS-VP64/MPH

When 293T cells grew to 60-70% confluence, 293T cells were infected simultaneously with two lentiviruses expressing dCAS-VP64/MPH vector (i.e., the two recombinant lentiviruses obtained in step 1). 2 days after viral infection, cells were first screened under pressure in medium containing 10. mu.g/ml Blastidin S (Selleck) for 7 days. Then, the cells were subjected to pressure screening in a medium containing 300. mu.g/ml of Hygromycin B (Selleck) for 7 days. The obtained positive cell clones were cultured in a medium containing 5. mu.g/ml of Blasticidin S and 150. mu.g/ml of Hygromycin B to maintain the expression of the transgene.

(V) sgRNA activates expression of endogenous Pdx1 gene

293T cells expressing dCAS-VP64/MPH at 3X 10⁵The density of/ml was seeded in 12-well plates, each well being seeded with 1ml of cells. The next dayAnd (5) transferring the sgRNA vector constructed in the step (three) and verified to be correct by sequencing into the cell by using a liposome. The cell transfection was divided into 6 groups, namely sgRNA 1-5 and sgRNA M (5 sgRNA mixed in equal mass ratio), and the amount of vector used in each group was 0.8 μ g (5 sgRNA vectors in the sgRNA M group, equal mass, 0.16 μ g each). 293T cells were used as transfection control.

When the synergistic effect of the sgRNA is detected, the cell transfection is divided into 5 groups, namely 0.8 mu g and 1.6 mu g of sgRNA3 vector, 0.8 mu g and 1.6 mu g of sgRNA4 vector and 1.6 mu g of sgRNA M (0.8 mu g of sgRNA3 vector and 0.8 mu g of sgRNA4 vector). 3 days after cell transfection, RT-PCR and qRCR are adopted to detect the change of the expression level of the Pdx1 gene, and the expression of the Pdx1 protein is confirmed by immunofluorescence analysis.

(six) RT-PCR and qPCR

Total RNA was isolated from the cells using TRIzol reagent and treated with DNase to remove genomic DNA contamination. Reverse transcription was performed using Superscript IV first-strand synthesis system (Invitrogen) using 1. mu.g total RNA as template. PCR amplification was performed using Taq DNA polymerase (Invitrogen) under the following conditions: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min, and 30 cycles; final extension 72 ℃ for 10 min. The qPCR reactions were performed using SYBR Green PCR Master Mix (AB), with each reaction repeated three times. Gene expression data were normalized to GAPDH transcript levels. Gene expression Change 2^-ΔΔCtAnd (4) a calculation method. The experimental results were confirmed by three independent biological replicates. The qPCR reaction conditions were as follows: pre-denaturation at 95 deg.C for 1 min; denaturation 95 ℃ for 5s, annealing 60 ℃ for 10s, elongation 72 ℃ for 15s, for 40 cycles.

Pdx1 primer:

5’-ATGAAGTCTACCAAAGCTCACGC-3’；

5’-TCTCTCGGTCAAGTTCAACATGA-3’。

GAPDH primer:

5’-CGAGATCCCTCCAAAATCAAGT-3’；

5’-TGAGGCTGTTGTCATACT TCTCAT-3’。

(VII) immunofluorescence assay

Cells were first fixed in paraformaldehyde for 30min, 4 ℃. Then, membrane rupture and blocking treatment were performed in PBS containing 0.1% Triton X-100 and 10% bovine serum. The primary antibody was then incubated overnight at 4 ℃ and finally with a fluorescently labeled secondary antibody at room temperature for 1 hr. The primary antibody used was anti-Pdx1(R & D Systems); the secondary antibody was donkey anti-coat AF594 (Invitrogen). Rat insulinoma INS-1 cells were used as a positive control. Nuclei were counterstained with DAPI.

(VIII) statistical analysis

All experiments were repeated at least 3 times. Results are mean ± sd. Statistical analysis using unpaired Student's t test, p <0.05 was significantly different.

Second, result in

1. sgRNA vector construction

The sgRNA oligonucleotide single strand is annealed to form a complementary oligonucleotide double strand. Subsequently, the double-stranded sgRNA was inserted into the BsmB I cleavage site of the lenti sgRNA (MS2) _ zeo backbone vector by ligation. After the ligation product is transformed by bacteria, positive clones are selected for sequencing identification, and finally a plasmid vector correctly inserted with 5 sgRNAs is obtained.

2. dCAS-VP64/MPH cell line

In order to efficiently and conveniently research the activation effect of sgRNA on endogenous Pdx1, the invention constructs a 293T cell line expressing dCAS-VP 64/MPH. The dCAS-VP64 and MPH vectors have Blasticidin and Hygromycin resistance genes, respectively. 2 days after infection of the cells with lentivirus expressing dCAS-VP64/MPH vector, the cells were first screened under pressure in medium containing 10. mu.g/ml of Blasticidin S. Control cells (293T cells not infected with virus) all died after 3-4 days of screening. After 5-7 days, the cells of the virus-infected group developed Blasticidin-resistant clones. Subsequently, the cells were subjected to pressure screening in a culture medium containing Hygromycin. After 3-4 days of screening, the cells in the control group all died. Part of the cell death of the virus-infected cells was also observed in the early stage of the screening, and the dead cells were mainly dCAS-VP64^-A cell. The positive cell clone formed after 5-7 days is dCAS-VP64⁺/MPH⁺A cell. The cells obtained were screened for further studies of endogenous gene activation expression.

3. sgRNA activates expression of Pdx1

After cells are transfected by the sgRNA for 3 days, total RNA of each group of cells is collected, and the expression condition of the Pdx1 gene is detected by RT-PCR. The results showed that the control 293T cells inherently expressed a certain amount of Pdx 1; compared with a 293T cell control group, the expression of the sgRNAs 1 to 5 and the sgRNA M group Pdx 1is obviously up-regulated, but the expression difference of Pdx1 between the groups cannot be distinguished (A in figure 2). To further analyze the expression difference of Pdx1 between groups, qPCR was used to detect the differential expression of Pdx 1. The results show that the expression of Pdx1 was significantly upregulated in each group compared to the control group, with the sgRNA 3and 4 groups being the most significant upregulation. synergy between sgrnas 1-5 (sgRNA M) was not evident (fig. 2B).

In order to further optimize expression of activating Pdx1 by the sgRNA, sgRNA 3and sgRNA4 are selected to observe the effect of the sgRNA 3and the sgRNA4 on gene activation. After 3 days after the sgRNA transfected cells, RT-PCR results showed that Pdx 1expression was significantly up-regulated in each group compared to the control group (a in fig. 3). The qPCR result shows that the activation effect of the sgRNA group with 1.6 mu g is better than that of the sgRNA group with 0.8 mu g, and certain dose dependence is achieved; the sgRNA M1.6 μ g group had better activation effect than the sgRNA 41.6 μ g, suggesting that there is a synergistic effect between sgRNA 3and sgRNA4 in activating expression of Pdx1 (fig. 3B).

To further confirm the expression of Pdx1 in cells at the protein level, sgRNA M group cells 3 days after transfection were collected, and cell slides were prepared for immunofluorescence analysis. The results showed that the 293T cell group Pdx1 reacted weakly positively (fig. 4), consistent with the PCR results (a and B in fig. 3). The INS-1 cell positive control group Pdx1 shows strong positive reaction; sgRNA M group cells also showed strong positive responses (fig. 4). The results show that the sgRNA can efficiently activate expression of Pdx1 in 293T cells.

The result of the invention shows that the expression of Pdx1 of 293T cells can be efficiently activated by applying CRISPR-on technology and performing chromatin remodeling. The invention plays an important role in the directional differentiation of the gene-induced PSCs into beta cells and the research on the embryonic development of pancreas.

<110> military medical research institute of military science institute of people's liberation force of China

<120> method for activating expression of endogenous Pdx1 gene based on chromatin remodeling

<130> GNCLN181726

<160> 5

<170> PatentIn version 3.5

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gcggagctgt caaagcgagc 20

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gttcagccgg gggccgtgat 20

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gcctggctgg ccgcactaag 20

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gcaggtgctc gcgggtacct 20

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gttttcgtga gcgcccattt 20

Claims

1. A method for activating the expression of an endogenous Pdx1 gene in a target cell is a method for activating the expression of the endogenous Pdx1 gene in the target cell based on a CRISPR-on SAM system;

the SAM system comprises sgRNA which targets the position from-400 bp to +1bp upstream of the transcription starting point of the Pdx1 gene; the target sequences of the sgRNAs are SEQ ID No. 3and SEQ ID No. 4.

2. The method of claim 1, wherein: the method comprises the following steps: allowing the target cell to express dCAS-VP64 fusion protein, MS2-P65-HSF1 fusion protein and the sgRNA, thereby activating endogenous Pdx1 gene expression in the target cell.

3. The method of claim 2, wherein: the method comprises the following steps:

4. The method of claim 3, wherein: in the step (1), when the recombinant lentivirus A is packaged, the adopted target plasmid is a lenti dCAS-VP64_ Blast vector; when the recombinant lentivirus B is packaged, the adopted target plasmid is MS2-P65-HSF1_ Hygro vector.

5. The method of claim 3, wherein: in the step (1), when the recombinant lentivirus A and the recombinant lentivirus B are packaged, the adopted packaging cells are 293T cells.

6. The method of claim 3, wherein: in the step (2), the vectors capable of expressing the sgRNA are a vector A and a vector B;

the vector A is a recombinant vector obtained by inserting a DNA fragment shown in SEQ ID No.3 into a lenti sgRNA (MS2) _ zeo backbone through a BsmB I enzyme cutting site; the vector B is a recombinant vector obtained by inserting a DNA fragment shown in SEQ ID No.4 into a lenti sgRNA (MS2) _ zeo backbone through a BsmB I enzyme cutting site.

7. The method according to any one of claims 1-6, wherein: the target cell is 293T cell or pluripotent stem cell.

8. A method for preparing a cell in which expression of an endogenous Pdx1 gene is activated, comprising the steps of: cells with activated expression of the endogenous Pdx1 gene prepared by the method of any one of claims 1-7.

9. The method of claim 8, wherein: the cell is 293T cell or pluripotent stem cell.

sgRNA, which is the sgRNA targeting the position from-400 to +1bp upstream of the transcription start point of the Pdx1 gene; the target sequences of the sgRNAs are SEQ ID No. 3and SEQ ID No. 4.

11. A vector capable of expressing the sgRNA of claim 10.

12. The carrier of claim 11, wherein: the carrier consists of a carrier A and a carrier B;

13. A vector set comprising the vector of claim 11 or 12, lenti dCAS-VP64_ Blast vector, MS2-P65-HSF1_ Hygro vector.

14. Use of the method of any one of claims 1 to 7 for inducing directed differentiation of pluripotent stem cells into islet beta cells.

15. Use of the method of any one of claims 1-7 to promote pancreatic embryonic development.