CN114085835A - Cancer cell analysis gene circuit assembly and preparation method thereof - Google Patents
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Abstract
The invention provides a cancer cell analysis gene circuit component and a preparation method thereof, belonging to the technical field of genetic engineering. The cancer cell analysis gene circuit component comprises a first identification gene circuit and a second identification gene circuit which are used for identifying the cancer cells in the same cancer cell state. microRNA sequences are inserted into the 3' UTR sections of the two genes, and the microRNAs come from cancer cells in the state of the cancer cells. The invention also discloses a preparation method of the cancer cell analysis gene circuit component. The invention can accurately mark the state of the cancer cells by obtaining the positive enhancer and the negative enhancer which can represent different phenotypes of the tumor cells so as to form the positive synthesized promoter and the negative synthesized promoter and combining the bar code region which can assist in judgment, can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, and is helpful for analyzing the heterogeneity among the cancer cells.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a cancer cell analysis gene circuit assembly and a preparation method thereof.
Background
With the continuous evolution of medical means and the rapid development of gene technology, medical workers find that the genetically modified cells can carry out high-specificity detection on specific states of other cells, such as cancer cells, for example, an artificially synthesized gene circuit which is established based on a specific microRNA expression mode in a HeLa cell and can distinguish a human cervical cancer cell HeLa from a human normal healthy cell HEK 293. The artificially synthesized gene circuits can play excellent roles in screening cancer cells and killing cancer cells in a targeted manner.
However, the driving element used in the existing gene circuit is often composed of a single fixed microRNA and an expression mode of a natural promoter, and the expression mode can only distinguish cancer cells from normal healthy cells, and can not distinguish various cancer cell subsets which appear in a total cancer cell population due to heterogeneity among the cancer cells, and the heterogeneity among the cancer cells is just the most critical factor of drug resistance recurrence of the cancer cells, so that accurate identification and judgment of the proportion of the cancer cells in various states which exist at the same time are helpful for researching the heterogeneity among the cancer cells. Therefore, it is desirable to provide a cancer cell analysis gene circuit module and a method for manufacturing the same, which can better identify the state of a cancer cell and determine the ratio of cancer cells in each state.
Disclosure of Invention
The present invention is directed to a cancer cell analysis gene circuit module and a method for manufacturing the same, which at least partially solve the above-mentioned problems.
According to one aspect of the present invention, there is provided a cancer cell analysis gene circuit assembly comprising at least two cancer cell analysis gene circuits, each of which is directed to a different cancer cell state and connected in parallel;
each cancer cell analysis gene circuit at least comprises a positive synthetic promoter, a barcode region and a negative synthetic promoter which are connected in sequence and correspond to the targeted cancer cell state, wherein the positive synthetic promoter comprises a positive enhancer possessed by the cancer cell in the cancer cell state, and the negative synthetic promoter comprises a negative enhancer possessed by the cancer cell in the cancer cell state.
Preferably, the forward synthetic promoter has a single protein binding region.
Preferably, the protein binding region of the negative synthetic promoter is single.
Preferably, the downstream end of the negative synthetic promoter further comprises a fluorescent protein sequence.
Preferably, the fluorescent protein sequence is an EYFP fluorescent protein sequence.
Preferably, the downstream end of the fluorescent protein sequence is also connected with a positive synthetic promoter, a barcode region and a negative synthetic promoter in sequence.
Preferably, each of the cancer cell analysis gene circuits comprises a 3'UTR segment, a fluorescent protein sequence, a Lac promoter, a positively synthesized promoter, a barcode region, a CMV promoter, an mScarlet sequence, a WPRE, a negatively synthesized promoter, a Lac terminator and a 3' UTR segment connected in sequence, and the positively synthesized promoter and the negatively synthesized promoter in each of the cancer cell analysis circuits comprise a positive enhancer and a negative enhancer from the cancer cell in the corresponding cancer cell state.
Preferably, the sequence of the barcode region is shown as SEQ ID NO. 1.
Preferably, the cancer cell states are at least two of a high invasive cell state, a cytoskeletal recombination active cell state, a high stem cell state, a high differentiated cell state, a fast division cycle cell state, a slow division cycle cell state, an immune escape cell state, and an anti-apoptotic cell state of MHCC97H line cancer cells.
The embodiment of the invention also provides a preparation method of the cancer cell analysis gene circuit, which comprises the following steps:
step 1: respectively obtaining a positive enhancer and a negative enhancer of the cancer cell in at least two cancer cell states;
step 2: obtaining a positively synthesized promoter and a negatively synthesized promoter based on the positively enhancer and the negatively enhancer for each of the cancer cell states;
and step 3: and (3) acquiring a barcode region, and sequentially connecting the positive synthetic promoter, the barcode region and the negative synthetic promoter to obtain the cancer cell analysis gene circuit. The invention can accurately mark the state of the cancer cells by obtaining the positive enhancer and the negative enhancer which can represent different phenotypes of the tumor cells so as to form the positive synthesized promoter and the negative synthesized promoter and combining the bar code region which can assist in judgment, can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, and is helpful for analyzing the heterogeneity among the cancer cells.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram of a method of obtaining a cancer cell analysis gene circuit according to the present invention;
FIG. 2 is a schematic diagram of a cancer cell analysis gene circuit according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. For convenience of description, only portions related to the invention are shown in the drawings.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The embodiment of the application provides a cancer cell analysis gene circuit component and a method for preparing the cancer cell analysis gene circuit, and it needs to be noted that the cancer cell analysis gene circuit in the gene circuit component can be applied to the identification of cancer cells in various states, such as various states of immune escape, skeleton recombination, invasion and migration and the like generated by an SMMC-7721 or an MHCC997H cancer cell line under a drug-resistant condition, and can also be combined with a targeted drug to prepare the targeted drug capable of accurately killing cancer cells of a certain subtype and the like, and the specific application of the targeted drug is not limited by the application. Accordingly, the cancer cell analysis gene circuit described in the embodiments of the present application, i.e., a gene segment composed of a promoter or an element including a promoter and having a recognition function, is called a gene circuit because it is responsible for recognizing a specific target gene segment and initiating a subsequent recognition display or treatment process.
The cancer cell line in the present embodiment mainly refers to a group of cancer cells with similar gene expression, such as SMMC-7721 or MHCC997H line, and the cancer cells in the present embodiment may be liver cancer, glioblastoma, biliary tract cancer, lung cancer, pancreatic cancer, melanoma, bone cancer, breast cancer, colorectal cancer, stomach cancer, prostate cancer, leukemia, uterine cancer, ovarian cancer, lymphoma or brain cancer, more preferably liver cancer, glioblastoma or biliary tract cancer, and most preferably liver cancer.
The cancer cell state in the present application mainly refers to various states of tumor cancer stem-stem maintenance differentiation, immune escape, skeleton recombination, invasion and migration, cell division cycle speed and the like, which are expressed by cancer cells under the condition of drug action or continuous culture, and the gene expression of the cancer cells is different from the cancer cell lines, especially, the enhancer in the gene sequence of the cancer cells under different cancer cell states is greatly different. Such cancer cells of different cancer cell states are often the cause of repeated recurrence during cancer therapy, and are also the main target of the present invention, because they have drug resistance and may have other malignant properties such as high metastasis.
In the examples of the present application, the viral vector used for testing and practicing the cancer cell analysis gene circuit component provided in the present invention may be derived from retrovirus, for example, Human Immunodeficiency Virus (HIV), Mouse Leukemia Virus (MLV), Avian sarcoma/leukemia virus (Avian sarcoma/leukemia virus, ASLV), Spleen Necrosis Virus (SNV), Rous Sarcoma Virus (RSV), Mouse Mammary Tumor Virus (MMTV), etc.), Adenovirus (Adenovirus), Adeno-associated virus (Adeno-associated virus, AAV), Herpes Simplex Virus (HSV), or the like, but is not limited thereto. The techniques for constructing promoters based on retroviruses for testing lentiviruses are well known to those skilled in the art and will not be described in detail herein.
In the present embodiment, the vector of the present invention includes a signal sequence or leader sequence for membrane targeting or secretion in addition to expression regulatory elements such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, and can be prepared in various ways according to the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector comprises a selectable marker for selecting host cells containing the vector, and when the expression vector is a replicable expression vector, a replicon may be included. The vector may be self-replicating or integrated into the host DNA.
In the present examples, the terms "linked" and "linking" are used to refer to the functional linkage of a nucleic acid expression control sequence that performs a conventional function to a nucleic acid sequence encoding a gene of interest. For example, when a promoter is inserted into a gene sequence of a retrovirus, the expression of the gene sequence of the retrovirus may be under the influence or control of the promoter. The "ligation" or "ligation" in the embodiments of the present application can be considered to be completed when the ligation property between the gene sequence of the promoter and the gene sequence of the retrovirus does not induce a frame shift mutation and the expression of the ribozyme is not inhibited by the expression regulatory sequence. This "joining" or "linking" process can be accomplished using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes well known in the art.
The fluorescent protein in the present embodiment may be selected from luciferase (luciferase), Green Fluorescent Protein (GFP), modified green fluorescent protein (mGFP), Enhanced Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), modified red fluorescent protein (mRFP), Enhanced Red Fluorescent Protein (ERFP), Blue Fluorescent Protein (BFP), Enhanced Blue Fluorescent Protein (EBFP), Yellow Fluorescent Protein (YFP), enhanced yellow fluorescent protein (ebp), cyan enhanced fluorescent protein (cfyfp), cyan fluorescent protein (ECFP), and the like, but is not limited thereto. By inserting a fluorescent protein into the gene of interest, the expression level of the cancer cell-specific ribozyme can be observed. It will be appreciated by those skilled in the art that this method can be used to determine whether a cancer cell is present in the presence of a particular type of cancer cell.
In the present embodiment, the enhancer may be obtained by a method described in a literature in which cancer cell lines of various cancer cell states have been already clarified, or by a method in which cancer cells of different cancer cell states are cultured by a combination of a drug-resistant culture or the like, and the enhancer contained in the cancer cells of different cancer cell states is analyzed and obtained therefrom.
The barcode regions (barcode regions) in the embodiments of the present application may be essentially any length and sequence suitable for uniquely identifying a gene type-associated feature. The barcode region can be used to represent any type of unique characteristic of a cancer cell, including the type of modification (e.g., DNA methylation, histone variants, etc.), the amount of replication (e.g., technical replication), or the concentration. The barcode region may also indicate the absence of any modification. In some embodiments, a barcode region has a length of about 6 to about 50 base pairs, such as about 7 to about 30 base pairs, for example about 8 to about 20 base pairs, such as about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs, or any range therein.
The sequence of one implementation mode of the sequence of the barcode region is shown as SEQ ID No.1, and specifically as follows:
gggcgagcgcgcgcaaatggcggcgactagtgatatccgtctctcggcgctgtgctcgttccacacaccgg。
the invention provides a cancer cell analysis gene circuit component, which comprises at least two cancer cell analysis gene circuits, wherein each cancer cell analysis gene circuit is respectively aimed at one cancer cell state. For example, for MHCC97H cancer cells, the cancer cell states that have been detected at present include 8 different cancer cell states, namely, highly invasive cells, cytoskeletal recombination active cells, highly dry cells, highly differentiated cells, fast-dividing periodic cells, slow-dividing periodic cells, immune escape cells, and anti-apoptotic cells, and of course, the cancer cell analysis genetic circuit in the embodiment of the present invention may be directed to all possible cell states of a certain cancer cell line, or may be directed to only some of the cancer cell states.
In an embodiment of the present application, the cancer cell analysis gene circuit includes at least a positive synthetic promoter, a barcode region, and a negative synthetic promoter, which are connected in this order and correspond to a targeted cancer cell state, the positive synthetic promoter including a positive enhancer possessed by a cancer cell in the cancer cell state, and the negative synthetic promoter including a negative enhancer possessed by a cancer cell in the cancer cell state. The positive enhancer and the negative enhancer are also called as a strong enhancer and a weak enhancer or a positive enhancer and a negative enhancer, the former has a positive enhancing effect on gene transcription, the latter has a negative regulating effect on gene transcription, and the combination of the two can complete a complete transcription control process. Since both the positive enhancer and the negative enhancer are derived from a cancer cell in a certain cancer cell state, the cancer cell in the cancer cell state can be accurately identified, and the technical effect of targeted identification can be achieved.
In the embodiment of the present application, the specific implementation manner of constructing the positive synthetic promoter and the negative synthetic promoter based on the positive enhancer and the negative enhancer may be based on the common knowledge in the art that the enhancer and the core sequence of the promoter are relatively close, and the positive synthetic promoter and the negative synthetic promoter including the corresponding enhancer may be prepared by using the technical means commonly used by those skilled in the art, such as overlapping the enhancer sequence with the CpG island sequence and performing protein modification. The present application is not particularly limited as to the specific manner of synthesizing a promoter in a positive direction and a promoter in a negative direction, and the means thereof are well known to those skilled in the art.
The cancer cell analysis gene circuit component in the present application mainly refers to a set including at least two different cancer cell analysis gene circuits, so that the component that integrates the cancer cell analysis gene circuits for a plurality of different cancer cell states can distinguish cancer cells in different states in one test.
The invention can accurately mark the state of the cancer cells by obtaining the positive enhancer and the negative enhancer which can represent different phenotypes of the tumor cells so as to form the positive synthesized promoter and the negative synthesized promoter and combining the bar code region which can assist in judgment, can analyze the proportion information of the cancer cells in various states by means of high-throughput cell experiments and the like, and is helpful for analyzing the heterogeneity among the cancer cells.
Preferably, the positively synthesized promoter may be configured to be single in the protein binding region, and the negatively synthesized promoter may also be configured to be single in the protein binding region. The term "protein binding region" used herein refers to a single promoter, including the above-mentioned positive synthetic promoter and negative synthetic promoter, whose sequence is a simple repeat, such as ATGCA, and the structure is ATGCA and ATGCA repeated multiple times, so that only 1 transcription factor protein is bound to the promoter, and no other proteins are bound to the promoter. Here, the binding sites of the positive synthetic promoter and the negative synthetic promoter that are constructed in advance may be queried and estimated based on the existing databases such as the JASPAR database, the NCBI database, and the like, and modified according to the query result, so that both the protein binding regions of the positive synthetic promoter and the negative synthetic promoter are set to be single.
Preferably, the cancer cell analysis gene circuit further comprises a fluorescent protein sequence. After the infection is completed, cancer cells which need to be further distinguished and counted subsequently can be roughly screened according to the fluorescence effect of the infected cancer cells, and the workload required for overall identification is reduced.
As a preferred implementation manner, as shown in FIG. 2, the fluorescent protein sequence, the positive synthetic promoter, the barcode region and the negative synthetic promoter are connected in sequence in the gene circuit for cancer cell analysis provided in the examples of the present application.
As shown in FIG. 2, the gene circuit for cancer cell analysis further comprises a Lac promoter and a Lac terminator, and the fluorescent protein sequence, the Lac promoter, the positive synthetic promoter, the barcode region, the negative synthetic promoter and the Lac terminator are sequentially connected. More specifically, the cancer cell analysis gene circuit provided in the examples of the present application includes a 3'UTR segment, a fluorescent protein sequence, a Lac promoter, a forward synthetic promoter, a barcode region, a CMV promoter, a mscarle sequence, a WPRE, a negative synthetic promoter, a Lac terminator, and a 3' UTR segment, which are sequentially linked.
According to another aspect of the present invention, as shown in FIG. 1, there is provided a method for preparing a cancer cell analysis gene circuit, comprising the steps of:
step 1: obtaining a positive enhancer and a negative enhancer of a cancer cell in at least two cancer cell states;
step 2: positively and negatively synthesized promoters are obtained based on the positive and negative enhancers corresponding to each cancer cell state.
And step 3: and (3) acquiring a bar code region, and sequentially connecting the positive enhancer, the bar code region and the negative enhancer to obtain a cancer cell analysis gene circuit. In step 3, the cancer cell analysis gene circuit further comprises a fluorescent protein sequence, and the fluorescent protein sequence, the positive synthetic promoter, the barcode region and the negative synthetic promoter are sequentially connected to obtain the cancer cell analysis gene circuit.
In step 1, the method for obtaining the enhancer in the present embodiment may be a method in which cancer cells of various cancer cell states are found, or a method in which cancer cells of different cancer cell states are cultured by means of combination drug-resistant culture or the like, and the enhancer contained in the cancer cells of different cancer cell states is analyzed and obtained.
In step 2, the specific implementation manner of constructing the positive synthetic promoter and the negative synthetic promoter based on the positive enhancer and the negative enhancer may be based on the common knowledge in the art that the enhancer and the core sequence of the promoter are relatively close, and the positive synthetic promoter and the negative synthetic promoter including the corresponding enhancer may be prepared by using the technical means commonly used by those skilled in the art, such as overlapping the enhancer sequence with the CpG island sequence and performing protein modification. The present application is not particularly limited as to the specific manner of synthesizing a promoter in a positive direction and a promoter in a negative direction, and the means thereof are well known to those skilled in the art.
Hereinafter, the present invention will be described in detail with reference to examples. However, the embodiments according to the present specification may be modified into various other forms, and the scope of the present specification should not be construed as being limited to the embodiments described below. The examples of this specification are provided to more fully describe the specification to those skilled in the art.
Examples
Material
Cell:
MHCC97H human highly metastatic hepatoma cell line provided by Shanghai institute of cell biology, Chinese academy of sciences.
Drugs and reagents:
doxorubicin (Doxorubicin), specification: 10mg, batch number: KFS276, supplier: (ii) Baiolaibo.
Sorafenib (Sorafenib) specification: 10mg, batch number: bay 43-9006, supplier: gonghai Ruihui chemical technology Co., Ltd.
Los Wei-1640 (RPMI-1640) Medium and Fetal Bovine Serum (FBS), purchased from GIBCO, USA.
Doxycycline (Doxycycline hydrochloride), specification: 10mM/mL, batch number: ID0390-10mM × 1ml (in water), supplier: solibao.
Plasmids were synthesized from general organisms (Anhui), the classifier plasmid backbone used the SWB-Blasticidin lentivirus backbone, and the synthetic promoter vector backbone used SWP-Puromycin.
Lipofectamine 3000, Specification: 1mL, batch number: l3000015, supplier: ThermalFisher.
Lentivirus packaging kit, specification: 100mL, batch number: GM-040801-: is filled with organisms.
The instrument comprises the following steps:
CX41 inverted phase contrast microscope and BX51 fluorescence microscope, both available from Olympus, Japan; DTX880 ELISA, available from Beckman, USA; 751GD ultraviolet spectrophotometer, available from Hangzhou Hull instruments, Inc.; CytoFLEX flow cytometer available from beckmann coulter international trade (shanghai) ltd.
The implementation method comprises the following steps:
1. 8 plasmids of 8 phenotypic cell state identifiers were mixed in equal mass ratio as target mixed plasmids, and the cell state identifier mixed plasmids were subjected to lentiviral packaging by co-transfection of HEK293T cells with 4 plasmids in 10cm cell culture dishes using a third generation lentiviral packaging system at a gag/pol: rev: vsv-g: target plasmid 5:2:3:8 transfection ratio. Wherein gag/pol is genome integration protein, rev is reverse transcription protein, and vsv-g is packaging protein.
2. After successful lentivirus packaging, the lentivirus supernatant was collected and filtered through a 0.45 μm filter to remove cells and debris.
Note: the filter suggests the use of cellulose acetate or Polyethersulfone (PES) filters (low protein binding) and no nitrocellulose filter can be used.
3. The lentivirus supernatant (4 parts) and the 5 Xlentivirus concentrate (1 part) were mixed at a volume ratio of 4:1, left at 4 ℃ for 2 hours or overnight, and initially mixed every 30min for 3 times.
Note: the longer the period of time at 4 ℃ (e.g., overnight), the higher the yield of virus may be; viral particles can be stored stably in a concentrate for several days, typically at 4 ℃.
4. Centrifuge at 4000g for 25min at 4 ℃.
5. The supernatant was carefully removed without vigorous shaking of the tube, and a white precipitate was generally visible (sometimes the precipitate was not visible).
6. The appropriate volume of DMEM (1/10 volume of the original supernatant) was added, carefully pipetted and the pellet resuspended.
7. The resuspended virus was dispensed in 50. mu.L tubes and stored in a freezer at 80 ℃.
Note: the frozen stock solution of lentivirus can not be frozen and thawed repeatedly, otherwise the virus titer can be reduced.
MHCC97H cells were infected with packaged lentiviruses in 8 wells of a six-well plate with lentiviruses of different cell status identifiers, infected and antibiotic-screened (Puromycin resistance screen, 2. mu.g/mL) for about 10 days. Then, 2 μ M RAF and VEGF targeting drug Sorafenib are used for adding drugs to MHCC97H cells which stably express identifiers of different cell states for 10 days, 20 days and 30 days, the cells expressing EYFP fluorescent protein (excitation light wavelength 513nm and emission light wavelength 527nm) are analyzed and sorted by flow cytometry, the genomes of the cells are extracted, amplification primers are arranged, next generation NGS 150bp single-end sequencing is carried out on the amplified fragments, the ratio of each phenotype is obtained by comparing bowtie 2 with a set phenotype representation barcode sequence and calculating the abundance of the barcode, and thus, the phenotype ratio data of various cell states are obtained: TABLE 1
Based on the above data, and the principle of maximum coverage of each subset, candidate promoters for sample 7, sample 8, and sample 9 were selected as driving elements for subsequent targeting of therapeutic gene circuits of multiple resistant subsets in the case of a three-promoter configuration; candidate promoters for sample 6, sample 7, sample 8, and sample 9 were selected as driving elements for subsequent targeting of therapeutic gene circuits of multiple resistance subpopulations in the case of a four promoter configuration.
According to the invention, a plurality of different preselected promoters are obtained by carrying out drug-resistant culture on cancer cells, the preselected promoters are used for constructing the promoter test lentivirus, and different cell phenotype state estimators are combined, so that a dual stable transgenic cancer cell line is obtained by infecting the cancer cells respectively by using the cell phenotype state estimator and the promoter test lentivirus, and a preselected promoter can be used for highly identifying various subtype cancer cell lines based on the drug-resistant culture result of the dual stable transgenic cancer cell line, so that the preselected promoter can be used as a driving element in a gene circuit constructed subsequently.
The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention herein disclosed is not limited to the particular combination of features described above, but also encompasses other arrangements formed by any combination of the above features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.
Sequence listing
<110> Zhuhaizhongke advanced technology research institute Co., Ltd
<120> cancer cell analysis gene circuit assembly and preparation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 71
<212> DNA
<213> Escherichia coli
<400> 1
gggcgagcgc gcgcaaatgg cggcgactag tgatatccgt ctctcggcgc tgtgctcgtt 60
ccacacaccg g 71
Claims (10)
1. A cancer cell analysis gene circuit assembly, comprising at least two cancer cell analysis gene circuits, each of which is directed to a different cancer cell state and connected in parallel;
each cancer cell analysis gene circuit at least comprises a positive synthetic promoter, a barcode region and a negative synthetic promoter which are connected in sequence and correspond to the targeted cancer cell state, wherein the positive synthetic promoter comprises a positive enhancer possessed by the cancer cell in the cancer cell state, and the negative synthetic promoter comprises a negative enhancer possessed by the cancer cell in the cancer cell state.
2. The cancer cell analysis genetic circuit assembly of claim 1, wherein the forward synthetic promoter has a single protein binding region.
3. The cancer cell analysis genetic circuit assembly of claim 1, wherein the protein binding region of the negative synthetic promoter is single.
4. The cancer cell analysis genetic circuit assembly of claim 1 further comprising a fluorescent protein sequence downstream of the negative synthetic promoter.
5. The cancer cell analysis genetic circuit assembly of claim 4, wherein the fluorescent protein sequence is an EYFP fluorescent protein sequence.
6. The cancer cell analysis gene circuit assembly of claim 4 or 5, wherein the downstream end of the fluorescent protein sequence is further connected with a positive synthetic promoter, a barcode region and a negative synthetic promoter in sequence.
7. The cancer cell analysis gene circuit assembly of claim 1 wherein each of the cancer cell analysis gene circuits comprises a 3'UTR segment, a fluorescent protein sequence, a Lac promoter, a positively synthesized promoter, a barcode region, a CMV promoter, an mScarlet sequence, a WPRE, a negatively synthesized promoter, a Lac terminator and a 3' UTR segment connected in sequence, and wherein the positively synthesized promoter and the negatively synthesized promoter in each of the cancer cell analysis circuits comprise a positive enhancer and a negative enhancer from a cancer cell in the corresponding cancer cell state.
8. The cancer cell analysis genetic circuit assembly of claim 1, wherein the barcode region has a sequence as set forth in SEQ ID No. 1.
9. The cancer cell analysis genetic circuit assembly of claim 1, wherein the cancer cell states are at least two of a highly invasive cell state, a cytoskeletal reorganization active cell state, a highly stem cell state, a highly differentiated cell state, a fast-division cycle cell state, a slow-division cycle cell state, an immune-escape cell state, and an anti-apoptotic cell state of MHCC97H line cancer cells.
10. The method of preparing a cancer cell analysis gene circuit according to any one of claims 1 to 9, comprising the steps of:
step 1: respectively obtaining a positive enhancer and a negative enhancer of the cancer cell in at least two cancer cell states;
step 2: obtaining a positively synthesized promoter and a negatively synthesized promoter based on the positively enhancer and the negatively enhancer for each of the cancer cell states;
and step 3: and (3) acquiring a barcode region, and sequentially connecting the positive synthetic promoter, the barcode region and the negative synthetic promoter to obtain the cancer cell analysis gene circuit.
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| CN114045264A (en) * | 2021-11-16 | 2022-02-15 | 珠海中科先进技术研究院有限公司 | Method for obtaining driving element |
| CN114075572A (en) * | 2021-11-16 | 2022-02-22 | 珠海中科先进技术研究院有限公司 | AND gate gene circuit and method for obtaining same |
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