Patent Application
20230102794
In accordance with 37 CFR § 1.831, the present specification makes reference to a Sequence Listing submitted electronically as a .xml file named “544837US_ST26.xml”. The .xml file was generated on Sep. 22, 2022 and is 51,342 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.
Embodiments described herein relate generally to a vector set for measuring transposase activity, a kit, a transposase activity measuring method, and a cell separation method.
In a technique of incorporating a nucleic acid into a genome of a cell, a transposase is used. The transposase is an enzyme having an activity of cutting out a DNA sequence in which a transposase recognition sequence is arranged at both ends, and an activity of inserting the cut-out DNA sequence into a transposase target sequence on a genome.
When such an enzyme is used or produced, it is important to confirm whether or not the enzyme works properly, that is, to confirm the activity of the enzyme in a necessary place. Therefore, a method for more easily confirming the activity of a transposase is required.
In general, according to one embodiment, a vector set includes a first vector and a second vector. The first vector includes a transposase target sequence, a first promoter sequence and a first reporter gene. The second vector includes 5′-side transposase recognition sequence, a 3′-side transposase recognition sequence, and a first enhancer sequence disposed between the two recognition sequences.
Embodiments will be described hereinafter with reference to the accompanying drawings. Note that, in these embodiments, substantially the same structural elements will be designated by the same reference symbols sign and the explanations therefor may be partly omitted. Further, the drawings are only schematic, and therefore, the relation between the thickness of each element and its planar dimension, the ratio in thickness between the elements and the like may be different from those of the actual cases.
According to embodiments, a vector for measuring the activity of a transposase is provided. The transposase to be subjected to activity measurement is, for example, a DNA-type transposase. The DNA-type transposase is, for example, but not limited to, PiggyBac, SleepingBeauty, Frog Prince, Hsma, Minos, Tol1, Tol2, Passport, hAT, Ac/Ds, PIF, Harbinger, Harbinger3-DR, Himarl, Hermes, Tc3, or Mos1. The transposase may be modified from the above-described transposase.
In the present specification, the activity of a transposase means a “cutting activity” for cutting out a sequence having a transposase recognition sequence at both ends from a nucleic acid sequence, and an “incorporating activity” for incorporating the cut-out sequence into a transposase target sequence on a genome.
A vector for measuring transposase incorporating activity (hereinafter, also referred to as a “first vector”) according to an embodiment is described below. As shown in part (a) of
The transposase target sequence 2 is a sequence as a target into which a transposase is to incorporate a DNA sequence. In other words, the transposase incorporates the cut-out DNA sequence into the transposase target sequence 2. The transposase target sequence 2 is, for example, a sequence including a plurality of TTAA sequences (T: thymine, A: adenine), and is preferably, for example, a sequence containing five TTAA sequences. Alternatively, a sequence including a plurality of TA sequences can also be used.
For example, the transposase target sequence 2 is preferably a base sequence shown in Table 1 below.
As is described in detail below, in a first embodiment, when the first vector 1 is used, a first enhancer sequence E1 can be incorporated into the transposase target sequence 2.
The first promoter sequence P1 is operably ligated to downstream of the transposase target sequence 2 so that gene activation on downstream of the first promoter sequence P1 is promoted when the first enhancer sequence E1 is incorporated into the transposase target sequence 2.
Herein, ligating encompasses a case where two sequences are ligated without other sequences being interposed between the two sequences, and a case where an arbitrary sequence is interposed between the two sequences. The arbitrary sequence is, for example, a spacer sequence. The spacer sequence is a nucleic acid sequence that is different from the sequences of the transposase target sequence 2, the first promoter sequence P1, the first reporter gene R1, the first transcription termination sequence T1, and their complementary sequences, and does not adversely affect the activity of these sequences.
The first promoter sequence P1 may be a base sequence of a known promoter capable of initiating transcription of a gene ligated downstream by its activity. The first promoter sequence P1 may be a promoter capable of expressing a gene by the presence of an enhancer. Alternatively, the first promoter sequence P1 may be a promoter having a low gene expression level when it is alone, but having a high gene expression level by the presence of an enhancer.
The first promoter sequence P1 is preferably, for example, a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) promoter, a thymidine kinase (TK) promoter, a ubiquitin (UbC) promoter, a human polypeptide chain elongation factor (EF1α) promoter, a hybrid (CAG) promoter of a cytomegalovirus enhancer and a chicken B-actin promoter, a mouse stem cell virus (MSCV) promoter, a Rous sarcoma virus (RSV) promoter, or the like. However, the first promoter sequence P1 is not limited to those listed above as long as it has a promoter function, and may be obtained by substituting or deleting any base in the base sequence of the promoter.
The first promoter sequence P1 is preferably a CMV promoter (SEQ ID NO: 2) having the base sequence shown in Table 2 or a promoter sequence (SEQ ID NO: 3) of the human polypeptide chain elongation factor gene (EF1α) shown in Table 3.
The first reporter gene R1 is operably ligated to downstream of the first promoter sequence P1 so that its gene expression is regulated by the activity of the first promoter sequence P1.
The first reporter gene R1 may be a base sequence of a gene encoding a known reporter protein. The first reporter gene R1 is, for example, a gene of a fluorescent protein such as a blue fluorescent protein gene, a green fluorescent protein gene, or a red fluorescent protein gene; a gene of luminescent enzyme proteins such as firefly luciferase gene, renilla luciferase gene or NanoLuc (registered trademark) luciferase gene; a gene of active oxygen generating enzymes such as xanthine oxidase genes or nitric oxide synthase genes; or a gene of a chromogenic enzyme protein such as a B-galactosidase gene or a chloramphenicol acetyltransferase gene. However, the first reporter gene R1 is not limited to the reporter genes listed above as long as the function as a reporter is not lost, and may be obtained by substitutina or deleting any base of the base sequence of the above-described reporter gene.
For example, as the first reporter gene R1, a firefly luciferase gene shown in Table 4, a luciferase gene derived from Oplophorus gracilirostris shown in Table 5, or the like can be used.
The first transcription termination sequence T1 is operably ligated to downstream of the first reporter gene R1 so as to terminate the transcription of the first reporter gene R1. The first transcription termination sequence T1 is, for example, a poly(A) addition signal sequence of simian virus 40 (SV40), a poly(A) addition signal sequence of a bovine growth hormone gene, an artificially synthesized poly(A) addition signal sequence, or the like. However, the first transcription termination sequence T1 is not limited thereto, and as long as it has a function as a transcription termination sequence, another sequence, a modified base sequence of the above-described transcription termination sequence, or the like may be used.
It is preferable to use a base sequence of a bovine growth hormone transcription termination sequence or a base sequence of a SV40 transcription termination sequence, which is shown in Table 6 and Table 7.
The first vector 1 is, for example, a circular double-stranded DNA molecule. The first vector 1 is, for example, a plasmid vector.
The first vector 1 may contain any base sequence in addition to the above sequence. Such a base sequence may be, for example, a base sequence having a specific function, or a sequence having no function.
The base sequence having a function is, for example, an additional reporter gene expression unit, a drug resistance gene, a replication initiation protein expression unit, and/or a replication initiation sequence.
The drug resistance gene can be used, for example, for screening of cells into which the vector has been introduced. As the drug resistance gene, for example, an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a blasticidin resistance gene, or the like can be used.
The replication initiation sequence is a sequence to which the replication initiation protein binds in order to initiate the replication of the first vector 1. When the first vector 1 is replicated, the reporter protein mass expressed from the first reporter gene R1 increases, and the activity of transposase can be measured with higher sensitivity. As the replication initiation sequence, for example, a replication initiation sequence derived from simian virus 40, Epstein-Barr virus, mouse polyomavirus, ColE1, or the like can be used.
The replication initiation protein may be originally present in a cell into which the first vector 1 is to be introduced, or may be introduced into the cell by a vector containing a replication initiation protein expression unit different from that of the first vector 1. The replication initiation protein may be expressed from the replication initiation protein expression unit, which may be provided in the first vector 1.
The transposase activity measuring method using the first vector 1, is performed by using a vector set containing any one of the above-described first vectors 1, and a second vector. The second vector is described below.
As shown in an example in part (b) of
These two recognition sequences are sequences that the transposase recognizes and binds to so as to cut out the sequence for being cut 4. The 5′-side transposase recognition sequence 4a and the 3′-side transposase recognition sequence 4b are, for example, inverted repeat sequences (IR), which include the same sequence in mutually opposite directions. The base sequence of the recognition sequence is selected according to the type of transposase whose activity is to be measured. Examples of the 5′-side transposase recognition sequence 4a and the 3′-side transposase recognition sequence 4b when the transposase is PiggyBac are shown in Tables 8 and 9 below, respectively.
An example of the base sequence of either one of the 5′-side transposase recognition sequence 4a and the 3′-side transposase recognition sequence 4b when the transposase is SleepingBeauty, is shown in Table 10 below. The other may include, for example, an inverted repeat sequence of the sequence shown below.
The first enhancer sequence E1 may be any known enhancer capable of promoting the activation of the first promoter sequence P1 and promoting the expression of a gene ligated to downstream thereof. The first enhancer sequence E1 can be selected according to, for example, the type of the first promoter sequence P1. As the first enhancer sequence E1, it is preferable to use, for example, a CMV enhancer, an SV40 enhancer, an RSV enhancer, a mouse retroviral terminal repeat (MLV LTR) enhancer, or the like. However, the first enhancer sequence E1 is not limited to the enhancer sequences listed above as long as the function as an enhancer is not lost, and may be obtained by substituting or deleting any base of the above-described enhancer sequence.
Table 11 shows an example of the base sequence of the enhancer sequence when the first promoter sequence P1 is a CMV promoter.
The second vector 3 may contain an arbitrary base sequence in addition to the above-described sequences. Such a base sequence may be, for example, a base sequence having a specific function, or a sequence having no function. The base sequence having a function is, for example, a reporter gene expression unit, a drug resistance gene, a replication initiation protein expression unit, and/or a replication initiation sequence.
The second vector 3 is, for example, a circular double-stranded DNA molecule. The second vector 3 is, for example, a plasmid vector.
A transposase activity measuring method using the first vector 1 and the second vector 3 is described below. The transposase activity measuring method includes, for example, the following steps shown in
An example of the method of the embodiment is described below in detail.
First, cells are prepared. The cells may be, for example, cells derived from humans, animals, or plants, or cells derived from microorganisms such as bacteria or fungi. The cells are preferably animal cells, more preferably mammalian cells, and most preferably human cells. The cells may be cells taken out of a living body, for example, cells separated from a body fluid such as blood, a tissue, a biopsy, or the like. The cells may be, for example, isolated cells, cultured cells, or established cells. Alternatively, the cells may be cells in a living body.
In a cell, there may be a transposase whose activity is to be measured. The transposase may be introduced, transcribed, and expressed in a cell, for example, in the form of a nucleic acid encoding it, for example, a DNA or an RNA. Alternatively, it may be in the form of a protein or a peptide introduced into a cell. Alternatively, it may be incorporated into the genome of a cell in advance, and expressed.
Next, the first vector 1 and the second vector 3 are introduced into the cell (introduction step S1). For example, when the cell is in a state of being taken out of a living body, the introduction step S1 can be performed by a known method such as a liposome method, a lipofection method, an electroporation method, a calcium phosphate co-precipitation method, a cationic polymer method, a microinjection method, a particle gun method, or a sonoporation method.
In particular, it is preferable to use a liposome method. In the liposome method, the first vector 1 and the second vector 3 are encapsulated in a liposome (lipid particle), and a composition or the like which contains it is brought into contact with a cell, so that, for example, the lipid particle is taken into the cell by endocytosis, and those encapsulated are released into the cell. Details of the lipid particles are described in the description of the following embodiment of a kit.
When the cell is a cell in a living body, the introduction can be performed by, for example, injecting or instilling a composition containing the first vector 1 and the second vector 3 into the living body. The composition may contain, for example, the lipid particles encapsulating the first vector 1 and the second vector 3.
When a transposase is introduced into a cell, the transposase may be introduced simultaneously with the introduction of the first vector 1 and the second vector 3, or either of these may be introduced earlier.
After the introduction step Si, for example, as shown in
The first signal 6 is a detectable signal obtained according to the type of the first reporter protein 5, and is, for example, fluorescence, chemiluminescence, bioluminescence, biochemiluminescence, coloration, or the like, or alternatively, presentation of a molecule such as a protein.
The first signal 6 is emitted from the first reporter protein 5 itself, or is generated by a reaction between the first reporter protein 5 and a specific substance (hereinafter, it is described as “first substance”), for example, an enzymatic reaction, binding, or the like. For example, when the first reporter protein 5 is an enzyme, the first substance is a substrate thereof. For example, when the first reporter protein 5 is luciferase, the first substance is luciferin.
Alternatively, the first signal 6 may be a signal derived from a further detection reagent (hereinafter, it is described as a “second substance”) for detecting the presence of a substance generated by a reaction between the first reporter protein 5 and a specific substance.
Next, the first reporter protein 5 is detected (the first detection step S2). The detection of the first reporter protein 5 can be performed, for example, by detecting the first signal 6. The detection may be performed by using any known method selected according to the type of the first reporter protein 5 or the first signal 6.
Detection can be performed, for example, in a living cell. However, it may be performed in an extract obtained by extracting the first reporter protein 5 from the cell.
For example, when the first substance and/or the second substance is used, these substances can be added to the cell at the beginning of the first detection step S2. These substances may be added to the culture medium for the cell or may be introduced into the cell. Alternatively, it may be added to a reporter protein extract obtained from the cell.
When the first reporter protein 5 is a fluorescent protein, the first signal 6 is obtained as fluorescence generated from the fluorescent protein by irradiating the cell with excitation light. The fluorescence (the first signal 6) can be detected by visual observation, a microscope, a flow cytometer, image analysis software, a fluorometer, or the like.
When the first reporter protein 5 is luciferase, luciferin is added thereto so that the first signal 6 is obtained as chemiluminescence. The chemiluminescence (the first signal 6) can be detected by visual observation, a microscope, a flow cytometer, image analysis software, a luminometer, or the like.
When the first reporter protein 5 is B-galactosidase, a substrate such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) or o-nitrophenyl-β-D-galactopyranoside (ONPG) is added, so that a first signal 6 is obtained as the absorbance of a cell solution or an extract. The absorbance (the first signal 6) can be detected by an absorptiometer, a spectrophotometer, a turbidimeter, or the like.
When the first reporter protein 5 is a nitric oxide synthase or a xanthine oxidase, active oxygen, which is generated by adding substrate, is obtained as the first signal 6. Active oxygen (the first signal 6) can be detected by an electron spin resonance apparatus (ESR apparatus) or the like.
When the first reporter protein 5 is a heavy metal binding protein, a heavy metal, which is bound to the reporter protein by adding detectable heavy metals, is obtained as the first signal 6. The heavy metal (the first signal 6) can be detected by a magnetic resonance imaging apparatus, a nuclear medicine diagnosis apparatus, an MRI imaging apparatus, or an X-ray computed tomography apparatus.
For example, since the intensity of the first signal 6 correlates with the expression level of the first reporter protein 5, the intensity of the expression of the first reporter protein 5 can be determined based on the intensity of the first signal 6. Alternatively, the first reporter protein 5 may be directly quantified.
Next, the activity of the transposase is evaluated from the result of detection of the first reporter protein 5 (the first evaluation step S3).
For example, when the first reporter protein 5 is highly expressed, it can be evaluated that the transposase TP at least has a good incorporating activity.
Here, when “the first reporter protein 5 is highly expressed” is described, it encompasses a case where the first reporter protein 5 (the first signal 6) is detected or its value equal to or more than a threshold value is obtained, a case where the expression level (the intensity of the first signal 6) of the first reporter protein 5 is increased, a case where the amount of an increase is equal to or more than a threshold value or more, and the like. When “having a good incorporating activity” is described, it encompasses having an incorporating activity and having a high incorporating activity.
When “increase” in the expression level (the intensity of the first signal 6) of the first reporter protein 5 is described, it encompasses, for example, an increase as compared to the value of the expression level (the intensity of the first signal 6) of the first reporter protein 5 before the first enhancer sequence E1 was incorporated into the first vector 1. The value of the expression level (the intensity of the first signal 6) of the first reporter protein 5 before the incorporation of the first enhancer sequence E1 may be 0, for example, depending on the type of promoter, or may be a smaller value than that after the incorporation of the first enhancer sequence E1. The value thereof before the incorporation of the first enhancer sequence E1 can be obtained, for example, by introducing the first vector 1 into a cell in advance, and performing detection before introducing the second vector 3 and/or the transposase TP. Alternatively, it may be a value obtained by the detection immediately after the first vector 1 and the second vector 3 (the transposase TP as necessary) are introduced, that is, before the cutting and the incorporation by the transposase TP are carried out. Alternatively, the intensity of the first signal 6 may be measured in advance in a cell into which first vector 1 is introduced and which does not contain the second vector 3 and/or the transposase TP, and the value may be used for comparison.
The threshold value may be, for example, a value of the expression level (the intensity of the first signal 6) of the first reporter protein 5, or the amount of an increase in the value, obtained when the measuring method of the embodiment is performed using a transposase TP that is known to have an incorporating activity.
In one embodiment, based on the expression level (the intensity of the first signal 6) of the first reporter protein 5, the degree of the incorporating activity of the transposase TP may be evaluated. The degree of incorporating activity is a ratio of transposases TP having an incorporating activity among the total transposases TP to be examined, or an amount of transposases TP having incorporating activity that are expressed or present in cells. For example, it is also possible to evaluate that the higher the expression level (the intensity of the first signal 6) of the first reporter protein 5, the greater the ratio or amount of the transposases TP having an incorporating activity.
Conversely, for example, when the first reporter protein 5 is poorly expressed, it can be evaluated that at least either the cutting activity or the incorporating activity of the transposases TP is poor.
Here, when “the first reporter protein 5 is poorly expressed” is described, it encompasses a case where the first reporter protein 5 (the first signal 6) is not detected or is less than the threshold value, or alternatively, a case where the expression level (the intensity of the first signal 6) of the first reporter protein 5 is not increased, a case where the amount of an increase is less than the threshold value, or a case where the amount of an increase is decreased.
When “having a poor activity” is described, it encompasses having no activity, and having a low activity.
As described above, the activity of the transposase TP can be evaluated by the first evaluation step S3. It is possible to at least find transposases TP with an incorporating activity.
The transposase activity measuring method may be performed using one device. The device includes, for example: a sample storage unit that stores cells; a liquid delivery unit that adds a composition containing the first vector 1 and the second vector 3, optionally the transposase TP, a first substance and/or a second substance used in the first detection step S2, and the like to the cells stored in the sample storage unit; a detection unit that detects the first signal 6 from the cells; an information processing unit including a program for calculating the presence or absence or the degree of the incorporating activity of the transposase TP from the information on the presence or absence or the intensity of the first signal 6 transmitted from the detection unit; and an output unit that outputs a result of calculation performed by the information processing unit.
According to the present method, it is not necessary, for example, to extract a nucleic acid and/or a reporter protein, thereby enabling to rapidly measure the activity of a transposase TP by a simple operation. In addition, it is possible to measure the activity of a transposase TP in a living cell without performing a step that may destroy a cell or denature or decompose a protein in a cell, such as extraction of a nucleic acid and/or a reporter protein. Therefore, the transposase TP and the cell whose activity is measured can also be used in an intact state for further steps.
The present method is performed, for example, on transposases whose activity is unknown regarding the presence or absence, or the degree thereof. For example, it may be used for measuring the activity of, for example, a self-synthesized transposase, a newly discovered or developed transposase, an existing transposase, or a transposase in DNA or RNA form or the like when the transposase is introduced into cells and/or expressed in cells, etc. For example, the present method can also be used for measurement of the activity of a commercially obtained transposase; measurement of the activity of a transposase in a particular process; measurement of the activity of a transposase in a novel process; or quality control of a product containing a transposase. Alternatively, when an introduction step using a transposase is performed as a part of a series of steps such as an experiment, it can also be used when it is desired to confirm whether or not these steps have been appropriately performed in order to proceed to the next step.
However, the application is not limited to these.
According to a further embodiment, a cell separation method using the first vector 1 is provided. The cell separation method is a method for separating cells based on the activity of transposase.
As shown in
(S11) an introduction step of introducing the first vector and the second vector into a cell;
The introduction step S11 and the first detection step S12 can be performed similarly to the introduction step S1 and the first detection step S2 of the transposase activity measuring method.
The first reporter gene R1 of the first vector 1 used in the cell separation method is preferably a gene that has low cytotoxicity and whose reporter protein can be detected in living cells.
In the separation step S13, for example, cells in which the first reporter protein 5 is highly expressed in the first detection step S12 are regarded as cells containing the transposase TP having at least good incorporating activity, and are separated from other cells. Alternatively, it is also preferable to evaluate the degree of an incorporating activity from the detection result so as to separate cells having particularly high incorporating activity.
The separation may be performed by any known means. For example, a flow cytometry technique such as a cell sorter can be used. Alternatively, desired cells may be manually separated while the cells are observed under a microscope. In that case, a fine probe or the like capable of sucking and discharging cells can be used.
As a result of the separation step S13, cells containing the transposase TP having at least a good incorporating activity can be accurately and easily separated. In addition, since cells can be separated in a living state, the separated cells can be used in further steps such as analysis.
According to a further embodiment, there is also provided a kit that can be used in the transposase activity measuring method and the cell separation method. The kit includes at least a vector set including the first vector 1 and the second vector 3.
The vector set is provided, for example, as a composition contained in a solvent. As the solvent, for example, endotoxin-free water, PBS, TE buffer, or HEPES buffer can be used. The composition may further contain an excipient, a stabilizer, a diluent, and/or an auxiliary.
The vector set may be included in the kit in a state of being contained in lipid particles. The lipid particle will be described with reference to
The material of the lipid membrane constituting the lipid particle 7 contains, for example, a phospholipid or a sphingolipid, such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, or cerebroside, or a combination thereof. In particular, it is preferable to contain 1,2-dioleoyl-3-trimethvlammonium propane (DOTAP) and/or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which adjusts the acid dissociation constant of the lipid particles 7.
Further, the material of the lipid particle 7 may contain: a biodegradable lipid (for example, compounds of Formula (1-01), Formula (1-02) and/or Formula (2-01) shown below, and the like); a lipid (for example, polyethylene glycol (PEG) dimvristoyl glycerol (DMG-PEG) or the like) which prevent the aggregation of the lipid particles 7; a component (for example, cholesterol or the like) that prevents leakage of an encapsulated substance from the lipid particles 7; a component for controlling the particle size of the lipid particles 7; a component for facilitating the fusion of the lipid particles 7 with the cells; and/or a component that facilitates the introduction of the encapsulated substance into the cells, or the like.
The lipid particle 7 may be a monomolecular membrane, a double membrane, a triple membrane, or the like. In addition, the lipid particle 7 may be formed of a single-layer membrane, or may be formed of a multi-layer membrane.
The lipid particle 7 may contain additional components as necessary, in addition to the first vector 1 and the second vector 3. The additional component is, for example, a pH adjusting agent and/or an osmotic pressure adjusting agent. The pH adjusting agent is, for example, organic acids such as citric acid and salts thereof, etc. The osmotic pressure adjusting agent is a sugar, an amino acid, or the like.
The lipid particle 7 can be produced using a known method used when a small molecule is enclosed in the lipid particle 7, for example, Bangam's method, an organic solvent extraction method, a surfactant removal method, a freeze-thaw method, or the like. For example, a lipid mixture of the material of the lipid particles 7 contained in an organic solvent such as alcohol at a desired ratio, and an aqueous buffer containing a component to be incorporated, such as a vector are prepared, and the aqueous buffer is added to the lipid mixture. The obtained mixture is stirred and suspended to form lipid particles 7 containing the vector and the like.
The kit may further contain a reagent for detecting the first reporter protein 5. The reagent is, for example, the first substance and/or the second substance described in the description of the first detection step S2.
The vector set and the reagent are provided in a container, individually or in combination of any components.
In a first embodiment described above, when the first reporter protein 5 (the first signal 6) is highly expressed, it can also mean that the sequence for being cut 4 has been normally cut out of the second vector 3. Thus, when the transposase TP is at least evaluated to have a good incorporating activity, it is also possible to expect that the transposase TP also has a cutting activity. In the description of a second embodiment below, a method for more accurately measuring the cutting activity simultaneously with the incorporating activity will be described.
In a second embodiment, a second vector further has a configuration capable of measuring the cutting activity of a transposase. As shown in part (a) of
Each sequence will be described below.
The second promoter sequence P2 is provided so that, with its activity, the transcription of the gene ligated to downstream can be started. As the second promoter sequence P2, any promoter sequence listed in the description of the first promoter sequence P1 can be used. The second promoter sequence P2 may be the same as or different sequence from the first promoter sequence P1.
The second reporter gene 5′-side fragment R2a and the second reporter gene 3′-side fragment R2b each include a 5′-side sequence and a 3′-side sequence obtained by bisecting a base sequence encoding one reporter gene (the second reporter gene R2, not shown).
The second reporter gene R2, as being bisected, is -41 -in a state in which its reporter activity is inactivated. The lengths of the base sequences of the second reporter gene 5′-side fragment R2a and the second reporter gene 3′-side fragment R2b, that is, the divisional positions, are not limited as long as they are selected so that the activity of the second reporter gene R2 is inactivated, and the lengths of the two sequences may be the same as or different from each other.
As the second reporter gene R2, any of the reporter genes listed in the description of the first reporter gene R1 can be used. The second reporter gene R2 is preferably selected as being different from the first reporter gene R1. For example, the first reporter gene R1 and the second reporter gene R2 may be luciferase genes having substrates with different luminescent colors, respectively, fluorescent protein genes having different fluorescent colors, or the like.
Examples of the base sequences of the second reporter gene 5′-side fragment R2a and the second reporter gene 3′-side fragment R2b when the second reporter gene R2 is a firefly luciferase gene (Table 4) are shown in the following Tables 12 and 13, respectively.
The sequence for being cut 4 is the same as that of the first embodiment described above, and includes a 5′-side transposase recognition sequence 4a, a 3′-side transposase recognition sequence 4b, and a first enhancer sequence E1 arranged between these two recognition sequences.
The second reporter gene R2 sequence formed by cutting out the sequence for being cut 4 and ligating the second reporter gene 5′-side fragment R2a and the second reporter gene 3′-side fragment R2b may include a sequence other than the sequence derived from the reporter gene as long as the function as a reporter is not lost. The other sequence is, for example, a sequence consisting of nucleotides that are a multiple of 3 in number, and is preferably a sequence encoding 0 to 20 amino acids. For example, a trace sequence remaining after the cutting may be present between the second reporter gene 5′-side fragment R2a and the second reporter gene 3′-side fragment R2b. Trace sequences may, but need not, encode amino acids.
The second transcription termination sequence T2 is operably ligated to downstream of the second reporter gene 3′-side fragment R2b so as to terminate the transcription of the second reporter gene R2. As the second transcription termination sequence T2, any of the transcription termination sequences listed in the description of the first transcription termination sequence T1 can be used. The second transcription termination sequence T2 may be the same sequence as or a different sequence from the first transcription termination sequence T1.
The second vector 8 may contain any other base sequence similarly to the second vector 3 of the first embodiment. Such a base sequence is, for example, a base sequence such as a reporter gene expression unit having a specific function, a drug resistance gene, a replication initiation protein expression unit, and/or a replication initiation sequence, or alternatively, a sequence having no function.
In a further embodiment, as shown in part (b) of
For example, when the second promoter sequence P2 enables the expression of a gene ligated to downstream by an enhancer, it is preferable to use the second enhancer sequence E2. However, when the second promoter sequence P2 is of a type that allows expression of a gene ligated to downstream thereof without an enhancer, it is not necessary to provide the second enhancer sequence E2.
According to second embodiment, there is provided a method for measuring the activity of a transposase in a cell by using a vector set including the first vector 1 described in the first embodiment and the second vector 8.
The transposase activity measuring method includes, for example, the following steps shown in
The introduction step S21 can be performed similarly to the introduction step S1 of the first embodiment except that the second vector 8 is used instead of the second vector 3.
The behavior of each vector after the introduction step S21 will be described with reference to
Thereafter, in the second vector 8, the second reporter gene 5′-side fragment R2a and the second reporter gene 3′-side fragment R2b are ligated to form the second reporter gene R2 (part (c) of
On the other hand, in the first vector 1, as in a first embodiment, the first enhancer sequence E1 is incorporated to form the first gene expression unit U1 (part (f) of
Thus, when the transposase TP has both a cutting activity and an incorporating activity, the transfer of the first enhancer sequence E1 contained in the sequence for being cut 4 from the second vector 8 to the first vector 1 causes the second signal 10 and the first signal 6 to be obtained from the second vector 8 and the first vector 1, respectively.
On the other hand, when the cutting activity of the transposase TP is poor, the sequence for being cut 4 is not cut out, and the second reporter gene R2 remains inactivated. As a result, the second reporter protein 9 is poorly expressed. In this case, since the sequence for being cut 4 is not cut out, the first reporter protein 5 can be poorly expressed regardless of whether the incorporating activity of the transposase TP is good or not.
In addition, in the case of a transposase TP having a good cutting activity and a poor incorporating activity, the second reporter protein 9 is highly expressed, but the first reporter protein 5 can be poorly expressed.
Next, a first detection step S22 is performed. The first detection step S22 can be performed similarly to the first detection step S2 of the first embodiment.
Next, the second reporter protein 9 is detected (the second detection step S23). The detection of the second reporter protein 9 can be performed, for example, by detecting the second signal 10. The detection may be performed by using the method described in the first detection step S2, which is selected according to the type of the second reporter protein 9.
Either the first detection step S22 or the second detection step S23 may be performed earlier, or the both may be performed simultaneously. When the both steps are performed simultaneously, it is preferable to select the first promoter sequence P1 and the second promoter sequence P2 (if necessary, the first enhancer sequence E1 and the second enhancer sequence E2) so that the expression level of the second reporter gene R2 is higher than the expression level of the first reporter gene R1. This is to prevent the second signal 10 from being buried in the first signal 6 and being difficult to detect.
Next, the cutting activity and incorporating activity of the transposase are evaluated from the results of the first detection step S22 and the second detection step S23 (the second evaluation step S24).
The incorporating activity can be determined, for example, from the expression of the first reporter protein 5 (the first signal 6) as the method described in the first evaluation step S3.
On the other hand, when the second reporter protein 9 is highly expressed (the intensity of the second signal 10 is high), it can be determined that the cutting activity of the transposase TP is good.
Meanwhile, when the expression level of the second reporter protein 9 (the intensity of the second signal 10) is low, it indicates that normal cutting has not been performed, so that it can be determined that the cutting activity is poor.
From the above, when the expression level of the first reporter protein 5 is high and the incorporating activity is good, and at the same time, when the expression level of the second reporter protein 9 is high, it can be determined that the cutting activity of the transposase TP is also good. When both the activities are good as described above, the transposase TP to be analyzed has a high efficiency of incorporating a nucleic acid into a genome, and can be suitable for use in applications such as genome editing.
When the expression level of the first reporter protein 5 is low and the incorporating activity is poor, it can be determined that the cutting activity is good if the expression level of the second reporter protein 9 is high. Conversely, if the expression level of the second reporter protein 9 is low, it can be determined that the cutting activity is also poor.
As described above, by using the second vector 8, it is possible to simultaneously evaluate the cutting activity and the incorporating activity of the transposase TP in the same cell. According to the present method, the incorporating activity is measured by using a sequence cut out in the measurement of the cutting activity. This is in line with the working mechanism of transposase in gene incorporation.
Therefore, according to the present method, it is possible to measure the activity of a transposase more accurately than separately measuring the cutting activity and the incorporating activity.
The first vector 1 and the second vector 8 of second embodiment can also be used in a cell separation method. The cell separation method includes the following steps, for example, as shown in
The introduction step S31, the first detection step S32, and the second detection step S33 can be performed similarly to the introduction step S21, the first detection step S22, and the second detection step S23 of the transposase activity measuring method.
In the separation step S34, desired cells are separated on the basis of, for example, the expression level of the first reporter protein 5 and/or the expression level of the second reporter protein 9. In particular, cells in which both the first reporter protein 5 and the second reporter protein 9 are highly expressed are preferably separated from other cells. As a result, cells containing a transposase TP having a good cutting activity and an incorporating activity are obtained. Alternatively, it is also preferable to evaluate the degree of each activity from the detection result to separate cells having particularly high activities in both of the activities.
According to this method, a cell containing a transposase TP having good activities in both respects is easily obtained. Therefore, for example, the efficiency of the genome-edited cell production using this cell can be improved.
The first vector 1 and the second vector 8 of second embodiment can also be provided as a kit similar to that of the first embodiment. Such a kit may further contain a reagent for detecting the second reporter protein 9 expressed from the second reporter gene R2.
In the first embodiment and the second embodiment, an example has been shown in which the first enhancer sequence E1 is transferred from the sequence for being cut 4 of the second vectors 3 and 8 to the first vector 1 by the transposase TP. However, the sequence to be transferred is not limited to the first enhancer sequence E1. For example, if the expression level (the intensity of the first signal 6) of the first reporter protein 5 changes before and after the transfer, the activity of the transposase TP can be evaluated regardless of which sequence is transferred. In a third embodiment, an example in which a sequence to be transferred is selected from sequences other than the first enhancer sequence E1 in the vector set of the first embodiment will be described.
The sequence to be transferred is a sequence involved in expression of a first reporter gene R1, the sequence being selected from the base sequences of the first vector 1 described in the first embodiment. The sequence involved in the expression of the first reporter gene R1 is selected, for example, from among the sequences included in a first gene expression unit U1, and is, for example, an entire sequence of a first enhancer sequence E1, a first promoter sequence P1, or the first reporter gene R1, or a partial sequence thereof.
The sequence involved in the expression of the first reporter gene R1 is preferably a base sequence having a length of about 50 to about 9000 bases, and more preferably a base sequence having a length of about 50 to about 2000 bases.
A first vector 1 according to third embodiment includes a sequence in which a sequence selected as involved in the expression of the first reporter gene R1 (for example, any one of or a part of El, P1, and R1 constituting the first gene expression unit U1) is substituted with a transposase target sequence 2. In other words, it has a configuration in which the transposase target sequence 2 is arranged instead of the sequence involved in the expression of the first reporter gene R1. In addition, a second vector 3 according to third embodiment has a configuration in which a sequence selected as a sequence involved in the expression of the first reporter gene R1 (for example, any one of or a part of E1, the first promoter sequence P1, and the first reporter gene R1 constituting the first gene expression unit U1) is arranged between a 5′-side transposase recognition sequence 4a and a 3′-side transposase recognition sequence 4b of the sequence for being cut 4.
Part (b) of
The second vector according to third embodiment may have the same configuration as that of the second vector 8 according to the second embodiment. Such a second vector includes, for example, a sequence selected as a sequence involved in expression of the first reporter gene R1 in place of the first enhancer sequence E1 of the second vector 8 or 8b shown in part (a) of
An example in which a sequence other than the first enhancer sequence E1 is selected as the sequence to be transferred is described above, but the sequence to be transferred is preferably the first enhancer sequence E1. For example, when the first promoter sequence P1 or the first reporter gene R1 is selected, the first reporter protein 5 is basically not expressed from the first vector 1 before the transfer. However, when the first enhancer sequence E1 is used, the first reporter protein 5 can be expressed even before transfer, and detecting that makes it possible to confirm the introduction of the first vector 1 into cells before analysis. Therefore, the first embodiment and the second embodiment using the first enhancer sequence E1 may be more preferable than a third embodiment.
The vector set of a third embodiment can be used in a kit, a transposase activity measuring method, and a cell separation method similar to those of the first embodiment and the second embodiment.
Hereinafter, examples in which vector sets of the embodiments are produced and used will be described.
Production of vector for measuring transposase cutting activity
First, an artificial DNA (SEQ ID NO: 14) shown in Table 14, in which a multi-cloning sequence was arranged between 5′-IR (SEQ ID NO: 8) and 3′-IR (SEQ ID NO: 9), which are recognition sequences of piggyBac, was synthesized (available from BEX Co., Ltd.).
Next, a vector (pCMV-Luc) in which a firefly luciferase expression unit including a cytomegalovirus (CMV) promoter sequence (SEQ ID NO: 2), a firefly luciferase gene derived from firefly (SEQ ID NO: 4), and a bovine growth hormone transcription termination sequence (SEQ ID NO: 6) was incorporated was prepared, and the artificial DNA (SEQ ID NO: 14) was incorporated therein such that the firefly luciferase gene was divided into two regions. The firefly luciferase gene was divided into a 5′-side region (SEQ ID NO: 12) and a 3′-side region (SEQ ID NO: 13). Thereby, a vector shown in Table 15 below: pCMV-LuIRuC (SEQ ID NO: 15) was obtained.
Next, an enhancer sequence (SEQ ID NO: 11) was incorporated into the multi-cloning sequence of the artificial DNA inserted into pCMV-LuIRuC. This enhancer sequence has a function of increasing the transcription level of a luciferase gene of a vector for transposase incorporating activity described in Example 2. As a result, a vector for measuring transposase cutting activity: pCMV-LuEuC (Table 16, SEQ ID NO: 16) shown in
Production of vector for measuring transposase incorporating activity
First, a vector (pEF1α-Luc) into which a luciferase expression unit including a promoter sequence (SEQ ID NO: 3) of a human polypeptide chain elongation factor gene (EF1α), a gene (SEQ ID NO: 5) of luciferase derived from Oplophorus gracilirostris (hereinafter, referred to as “OG luciferase”), and a bovine growth hormone transcription termination sequence (SEQ ID NO: 6) was incorporated was prepared. An artificial DNA (SEQ ID NO: 14) in which TTAA as a target sequence of piggyBac was repeated 5 times was incorporated into an upstream region of an EF1α promoter of pEF1α-Luc. As a result, a vector for measuring transposase incorporating activity: pTTAAx5-EF1α-Luc (Table 17, SEQ ID NO: 17) shown in
Production of piggyBac mRNA
The piggyBac mRNA was synthesized using pGEM-GL-PB4 (available from BEX Co., Ltd.) as a template. CUGA (registered trademark) 7 in vitro transcription kit (NIPPON GENE Co., Ltd.) was used for synthesis. A Cap structure and a poly(A) structure were added to a untranslated region (UTR) and a 3′-UTR of RNA (Table 18, SEQ ID NO: 18) obtained by purifying this, respectively, so that it became an mRNA.
The addition of the Cap structure and the poly(A) structure was performed with mScript™ mRNA Production System (available from CellScript, LLC.). The operations of the above experiments followed the protocol of each kit.
Production of HyperpiggyBac mRNA
HyperpigyBac mRNA was synthesized using pGEM-GL-TS-HyPB (available from BEX Co., Ltd.) as a template. CUGA (registered trademark) 7 in vitro transcription kit (NIPPON GENE Co., Ltd.) was used for synthesis. A Cap structure and a poly(A) structure were added to a 5′ untranslated region (UTR) and a 3′-UTR of RNA (Table 19, SEQ ID NO: 19) obtained by purifying this, respectively, so that it became an mRNA.
The addition of the Cap structure and the poly(A) structure was performed with mScript™ mRNA Production System (available from CellScript, LLC.). The operations of the above experiments followed the protocol of each kit.
Measurement of piggyBac activity by pCMV-LuEuC and pTTAAx5-EF1α-Luc
Human T-cell leukemia cells (Jurkat, manufactured by ATCC (registered trademark)) cultured in a TexMACS medium (manufactured by Miltenyi Biotec) were collected by centrifugation, and then seeded on a 96-well plate at 5.0×105 cells/well. A vector for measuring transposase cutting activity (pCMV-LuEuC) produced in Example 1, a vector for measuring transposase incorporating activity (pTTAAx5-EF1α-Luc) produced in Example 2, and piggyBac mRNA prepared in Example 3 were encapsulated in lipid nanoparticles containing a cationic lipid, and the lipid nanoparticles were added to the wells so that the amounts of the vectors and mRNA to be added were as shown in Table 20. Thereafter, the culture plate was housed in an incubator, and the cells were cultured at 37° C. in a 5% CO2 atmosphere.
After 48 hours from the addition of the lipid nanoparticles, the culture plate was taken out of the incubator, and 50 μL of a culture solution was collected in a 96 well plate. Using ONE-Glo™ Luciferase Assay System (available from Promega Corporation) and Nano-Glo (registered trademark) Luciferase Assay System (available from Promega Corporation), the luminescence intensities of firefly luciferase expressed from a firefly luciferase gene and OG luciferase expressed from an OG luciferase gene were measured with a luminometer (Infinite (registered trademark) F200PRO, available from Tecan Corporation). The measurement was performed according to the manuals attached to the kit and the luminometer.
The results of
Measurement of piggyBac activity and HyperpiggyBac activity by pCMV-LuEuC and pTTAAx5-EF1α-Luc
Human T-cell leukemia cells (Jurkat, manufactured by ATCC (registered trademark)) cultured in a TexMACS medium (manufactured by Miltenyi Biotec) were collected by centrifugation, and then seeded on a 96-well plate at 2.0×105 cells/well. A vector for measuring transposase cutting activity (pCMV-LuEuC) produced in Example 1, a vector for measuring transposase incorporating activity (pTTAAx5-EF1α-Luc) produced in Example 2, piggyBac mRNA prepared in Example 3, and HyperpiggyBac mRNA produced in Example 4 were encapsulated in lipid nanoparticles containing a cationic lipid, and the lipid nanoparticles were added to the wells so that the amounts of the vectors and mRNA to be added were as shown in Table 21.
Thereafter, the culture plate was housed in an incubator, and the cells were cultured at 37° C. in a 5% Co2 atmosphere.
After 48 hours from the addition of the lipid nanoparticles, the culture plate was taken out of the incubator, and 50 μL of a culture solution was collected in a 96 well plate. Using ONE-Glo™ Luciferase Assay System (available from Promega Corporation) and Nano-Glo (registered trademark) Luciferase Assay System (available from Promega Corporation), the luminescence intensities of firefly luciferase expressed from a firefly luciferase gene and OG luciferase expressed from an OG luciferase gene were measured with a luminometer (Infinite (registered trademark) F200PRO, available from Tecan Corporation). The measurement was performed according to the manuals attached to the kit and the luminometer.
The results of
While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-003669 | Jan 2021 | JP | national |
This application is a Continuation Application of PCT Application No. PCT/JP2021/033368 filed Sep. 10, 2021 and based upon and claiming the benefit of priority from Japanese Patent Application No. 2021-003669, filed Jan. 13, 2021, the entire contents of all of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2021/033368 | Sep 2021 | US |
| Child | 17930961 | US |
