Cell for expressing labeled PERK as well as construction method and application thereof

Abstract

The invention relates to a method for constructing a cell expressing PERK with a tag. The method comprises the step of knocking nucleic acid for coding the tag into PERK by using a Crispr / Cas9 system. The Crispr / Cas9 system comprises a guide RNA (Ribonucleic Acid) and a repair structure, wherein the repair structure comprises a segment targeted by the guide RNA and a tag. According to the invention, a HA tag is knocked into a U20S cell line by using a Crispr / Cas9 system, so that the C end of PERK is provided with a hemagglutinin tag. By using the cell, interaction, physiological functions, drug screening and the like of PERK can be researched by observing endogenous PERK behaviors, and the cell plays an important role in researching PERK signal pathways in pathophysiology and disease progression of neurodegenerative diseases, myelin disorder, tumor growth and cancers.

Description

Technical Field

[0001] The invention belongs to the field of endoplasmic reticulum disease research, and specifically relates to cells expressing labeled PERK and a construction method and application thereof. Background Art

[0002] In the ER stress-induced unfolded protein response (UPR), the protein kinase RNA-like endoplasmic reticulum kinase (PERK), encoded by EIF2AK3, acts as a key regulator. A series of cellular stress conditions may trigger the disruption of endoplasmic reticulum (ER) balance and lead to ER stress response, further activating the unfolded protein response (UPR) branch. This leads to the subsequent phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of phosphorylation of eukaryotic initiation factor 2α (eIF2α), and rapid downregulation of intracellular protein synthesis and gene translation. The accumulation and aggregation of disease-specific mis / unfolded proteins are associated with a variety of neurodegenerative diseases, including multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and cancer.

[0003] In addition, chronic activation of the UPR may induce paradoxical responses by initiating apoptosis. Multiple lines of evidence suggest that PERK / eIF2α / ATF4 / CHOP play a key role in tumor progression.

[0004] In order to study the physiological and pathological functions of the PERK signaling pathway and screen drugs in PERK-related diseases, it is necessary to construct cells expressing tagged PERK. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for constructing a cell expressing tagged PERK, comprising the step of knocking in a nucleic acid encoding a tag into PERK using a Cr i spr / Cas9 system.

[0006] In a specific embodiment, the Cr i spr / Cas9 system includes a guide RNA and a repair structure, wherein the repair structure includes a segment targeted by the guide RNA and a tag.

[0007] In a specific embodiment, the sequence of the guide RNA is selected from SEQ ID NO: 1-3.

[0008] In a specific embodiment, the nucleic acid sequence of the segment targeted by the guide RNA is shown in SEQ ID NO:10.

[0009] In a specific embodiment, the tag is a hemagglutinin tag.

[0010] In a specific embodiment, the tag is knocked into the 3' end of the gene encoding PERK as determined by sequencing.

[0011] The present invention also provides cells expressing tagged PERK constructed by the above method.

[0012] The present invention also provides the use of the above cells in the functional study of PERK or drug screening.

[0013] The present invention uses the Cr i spr / Cas9 system to knock in the HA tag in the U20S cell line, so that the C-terminus of PERK carries a hemagglutinin tag. Using this cell, we can study PERK interactions, physiological functions, drug screening, etc. by observing the behavior of endogenous PERK, which plays an important role in studying the pathophysiology and disease progression of PERK signaling pathways in neurodegenerative diseases, myelin disorders, tumor growth and cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the pSpCas9(BB)-2A-GFP(PX458) plasmid map.

[0015] Figure 2 The results of the PCR reaction products of the guide RNA and plasmid constructs in E. coli were separated and visualized on agarose gel.

[0016] Figure 3 Schematic diagram of the pcDNA5 / FRT / TO V5 plasmid map.

[0017] Figure 4 The PERK_HA portion was amplified by PCR and the amplified products were separated on 3% agarose gel.

[0018] Figure 5 This is the sequencing alignment result of PERK_HA cell line. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0020] 1. Design of guide RNA for knocking out human PERK

[0021] Based on the human PERK gene sequence and its surrounding sequences, three guide RNAs (guide 1-3, sequences as shown in SEQ ID NO: 1-3, respectively) of the human PERK gene for Cr i spr Cas9 knockout of PERK were designed, and the following oligonucleotides were synthesized, as shown in Table 1.

[0022] Table 1 Guide RNA sequences

[0023] Guide RNA name sequence guide1-hperk-kinF cacc gATTGCTTGGCAAAGGGCTAT(SEQ ID NO:4) guide1-hperk-kinR aaac ATAGCCCTTTTGCCAAGCAATc(SEQ ID NO:5) guide2-hperk-kinF cacc gACAACTTAAGGCTAATTGCT(SEQ ID NO:6) guide2-hperk-kinR aaac AGCAATTAGCCTTAAGTTGTc(SEQ ID NO:7) guide3-hperk-kinF cacc gTAAGGCTAATTGCTTGGCAA(SEQ ID NO:8) guide3-hperk-kinR aaacTTGCCAAGCAATTAGCCTTAc(SEQ ID NO:9)

[0024] 2. Construction of Guide RNA Plasmid

[0025] The three pairs of oligonucleotides were annealed to obtain three sticky-end fragments, which were ligated to the pSpCas9(BB)-2A-GFP(PX458) plasmid digested with BbsI. Figure 1 ) to obtain the guide RNA plasmid, which was used to transform Escherichia coli.

[0026] Positive transformants were screened by PCR and sequencing. Figure 2 As shown, multiple positive transformants were screened for each guide RNA plasmid.

[0027] 3. Construction of Repair PERK_HA Plasmid Complex

[0028] The PERK region and HA fragment targeted by the guide RNA were amplified using primers, ligated, and cloned into the pcDNA5 / FRT / TO V5 plasmid ( Figure 3 ) to obtain the CAS9 knockout plasmid for repairing PERK.

[0029] The primer pair sequences for amplifying the PERK region are shown in SEQ ID NOs: 10 and 11; the primer pair sequences for amplifying the HA fragment are shown in SEQ ID NOs: 12 and 13.

[0030] 4. Nucleofection of sgRNA, repair constructs, and Cas9 nuclease

[0031] The steps for nucleofection are as follows:

[0032] U20S cells were passaged 2 days before the nuclear transfection experiment, preferably when the cells reached 80% confluence.

[0033] For each sample, 2 μg of plasmid DNA was prepared by mixing 18.2 μl of plasmid solvent into 81.8 μl of Lonza nucleofection solution and mixing gently.

[0034] Add 1 ml of cell culture medium to each well of a 6-well plate and place the plate in an incubator at 37 degrees with 5% CO2.

[0035] Remove the 75 cm2 culture flask and wash the cells once with 10 ml PBS. Add PBS / EDTA to the cells and wait for the cells to be suspended.

[0036] The cells were harvested and counted, and 1×10 6 The cells were transfected once, centrifuged at 90×g for 10 min at room temperature, and the supernatant was completely discarded.

[0037] Add 10 μl of nucleofection solution to 1×10 6 cells, and added 2 μg PX458_gRNA guide RNA plasmid DNA (2 μl) and 1 μg pCDNA_PERKHA repair plasmid DNA.

[0038] The suspended cell solution to which the guide RNA plasmid and PERKHA repair plasmid have been added is transferred to a cuvette and then electroporated.

[0039] Immediately add 500 μl of cell culture medium to the electro-transfected cell solution and transfer to a 75 cm 2 culture flask, and culture in an incubator at 37° C. and 5% CO 2 .

[0040] After the culture, the cells were harvested and suspended in 700 μl of PBS + 2% FCS, and passed through a 40 μm cell strainer. The cells with GFP single cells were sorted into two 96-well plates, 100 μl of conditioned medium was added to each well, and then sent to the sorting cell instrument ASTRIOS EQ cell sorter.

[0041] 5. Single cell clone screening

[0042] 1) Wash the cells thoroughly with 1× PBS and add about 1.5 ml of trypsin;

[0043] 2) Return to the incubator for ~5 minutes, add an equal amount of culture medium, collect cells and centrifuge at 1250 rpm for 4 minutes, and remove the supernatant;

[0044] 3) Resuspend the cells in 5 ml of cell solution and count the cells;

[0045] 4) 50 cells were cultured in three 10 cm culture dishes, and another 100 cells were cultured in three 10 cm culture dishes;

[0046] 5) After 2-3 days, single cell clones were selected and transferred to 96-well plates for culture. Approximately 60 single cell clones were selected from each culture dish.

[0047] 6) Place the 96-well plate in an incubator for 2-3 weeks to allow the single cells to grow into large clones, and replace the cell medium every 5-6 days.

[0048] 6. Cell genotype screening

[0049] 1) Extraction of cell genomic DNA:

[0050] Prepare DNA extraction reagent (1 ml Direct PCR Lysis Reagent + 40 μl Proteinase K - enough for 20 samples)

[0051] a. Add 50 μl of DNA isolation reagent to each PCR tube containing approximately 20 μl of cell suspension;

[0052] b. Vortex for 10 seconds, then pulse spin;

[0053] c. Incubate at 56°C and 300 rpm for 16 hours;

[0054] d. Incubate at 95°C for 10 minutes.

[0055] 2) Genomic DNA isolated from clones was PCR amplified using Dream Taq polymerase (PCR reaction conditions: 96°C, 5 minutes; (96°C, 45 seconds; 70°C, 20 seconds; 72°C, 1 minute), 7 cycles; 72°C, 5 minutes), and PCR products were separated on a 3% agarose gel prepared in 1× TBE. The resolution of this gel enables identification of small insertions and deletions in the genomic region of interest that may disrupt the transcript reading frame.

[0056] The results are as follows Figure 4 As shown, from the PCR product, cell line 49 can be considered to have correctly inserted the HA fragment and can become a positive clone cell.

[0057] 3) Sequencing was performed on the positive clone cell line 49. The sequencing results are as follows Figure 5 , the sequence alignment was completely correct, and HA was successfully added behind PERK.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for constructing a cell expressing tagged PERK, characterized in that: The method comprises the steps of knocking in a nucleic acid encoding a tag into PERK using the Crispr / Cas9 system.

2. The method according to claim 1, characterized in that The Crispr / Cas9 system includes a guide RNA and a repair structure, wherein the repair structure includes a segment targeted by the guide RNA and a tag.

3. The method according to claim 2, characterized in that The sequence of the guide RNA is selected from SEQ ID NO: 1-3.

4. The method according to claim 2, characterized in that: The nucleic acid sequence of the segment targeted by the guide RNA is shown in SEQ ID NO:

10.

5. The method according to claim 2, characterized in that: The tag is a hemagglutinin tag.

6. The method according to claim 1, characterized in that Sequencing confirmed that the tag was knocked into the 3' end of the gene encoding PERK.

7. A cell expressing tagged PERK, characterized in that The method is constructed by any one of claims 1 to 6.

8. Use of the cell according to claim 7 in functional study or drug screening of PERK.

Images