WO2024090563A1 - Nucleic acid for gene expression uses, gene expression vector, method for producing gene expression vector, and gene expression method - Google Patents

Abstract

The purpose of the present invention is to provide: a nucleic acid for gene expression uses, which can amplify a conditional gene expression vector stably, can be used in a gene expression analysis in a wide variety of organism species and cell species, can be predicted with respect to the expression amount thereof, and can be used in a gene expression vector having broad utility and capable of being used for an analysis at an organism individual level; and a gene expression vector, a method for producing the gene expression vector, and a gene expression method, in each of which the nucleic acid is used. Provided are: a nucleic acid for gene expression uses, which is intended to be use in a gene expression vector containing a recombinant enzyme expression gene, is introduced to the upstream of the recombinant enzyme expression gene, and comprises a sequence in which TA is repeated at least five times; and a gene expression vector, a method for producing the gene expression vector, and a gene expression method, in each of which the nucleic acid is used.

Description

Nucleic acid for gene expression, gene expression vector, method for producing gene expression vector, and gene expression method

The present invention relates to the expression of a specific gene in an organism, in particular to a nucleic acid contained in a gene expression vector when expressing a specific gene under certain conditions, a gene expression vector using the same, a method for producing a gene expression vector, and a gene expression method.
This application claims priority based on Japanese Patent Application No. 2022-173479, filed on October 28, 2022, the contents of which are incorporated herein by reference.

Systems for recombinant genes to express or knock out specific genes have a wide range of applications, and are widely used, for example, in analyzing gene functions in living organisms.
For example, the Cre-LoxP system is widely used because it is possible to knock out a gene with a specific sequence flanked by LoxP sequences by expressing the Cre gene.

The Cre-LoxP system can be used for conditional knockout by expressing the Cre gene under certain conditions. For example, by expressing the Cre gene under certain conditions, it is possible to knock out the gene of the specific sequence under those conditions. For example, such conditions can be achieved by introducing a tissue-specific promoter upstream of the Cre recombinase gene of Cre-LoxP, which causes the Cre gene to be expressed only in specific tissues, thereby making it possible to knock out the gene of the specific sequence under the condition of a specific tissue.

For example, Patent Document 1 discloses a non-human mammalian animal that retains at least one human cytochrome P450 gene, the expression of which is inducible by a compound that serves as a substrate for the gene product, as an example of gene introduction and knockout technology using the Cre-LoxP system, and also discloses a technology for disrupting the cytochrome P450 gene specific to the non-human mammalian animal using the Cre-loxP system. This technology aims to create a mouse with a human P450 gene (CYP3A family) and also with the mouse's endogenous P450 gene (Cyp3a family) disrupted, a so-called gene replacement mouse.

Non-Patent Document 1 discloses a technology that utilizes the CREM (Cre-M) sequence, in which an intron sequence from the human β-globin gene is inserted into the Cre gene. This technology inserts an intron sequence into the recombinase gene, preventing the recombinase from being expressed in E. coli and making it possible to amplify the vector gene in E. coli.

WO 01/011951

　Conventionally, when creating a Cre-LoxP mouse into which the Cre-LoxP gene has been introduced, a procedure that involves at least two generations of mice has been used. For example, a Cre mouse into which a Cre vector containing a Cre recombinase sequence has been introduced, and a LoxP mouse into which a LoxP vector containing a LoxP sequence and a specific sequence sandwiched between the LoxP sequences has been introduced are created. The Cre mouse and the LoxP mouse are then crossed to create a Cre-LoxP mouse into which the Cre vector and the LoxP vector have each been introduced.

However, this method of producing Cre-LoxP mice requires at least one crossbreeding, which is costly and time-consuming. Also, although mice are one of the organisms with a relatively short generational turnover period, it was thought that producing Cre-LoxP organisms, for example, in research on organisms with even longer generational turnover periods, would require even greater cost and time.

Therefore, the present inventors investigated the possibility of further simplifying the production of Cre-LoxP organisms and broadening the range of applications of the Cre-LoxP system by introducing a gene containing a Cre recombinase sequence, a LoxP sequence, and a specific sequence sandwiched between LoxP sequences into a target organism.
In other words, we designed a vector such as a plasmid having both the Cre vector sequence and the LoxP vector sequence, and attempted to introduce the Cre-LoxP vector into the organism into which it was introduced (first generation) in order to study gene expression and knockout using the Cre-LoxP system.

However, when gene transfer was attempted using the Cre-LoxP vector, it was found to be ineffective, i.e., when the nucleic acid of the Cre-LoxP vector was prepared, propagated in E. coli or the like, and purified, it was found that the gene of the specific sequence flanked by the LoxP sequences had disappeared.
It was believed that the disappearance of the gene with this specific sequence was due to the Cre-LoxP system functioning in E. coli during the proliferation of the nucleic acid. That is, usually, after a nucleic acid that will become a vector gene is prepared, the nucleic acid containing the vector gene is propagated using E. coli or the like. However, it was believed that at this point, Cre is expressed and functions in E. coli, thereby exerting its function of removing the gene with the specific sequence sandwiched between the LoxP sequences (spontaneous recombination).
In this Cre-LoxP vector, a tissue-specific promoter that does not express Cre except in certain tissues is inserted upstream of the Cre recombinase. However, it is believed that Cre is expressed in E. coli regardless of whether the promoter is tissue-specific or not, and spontaneous recombination occurs.
From these results, it was difficult to use the Cre-LoxP vector having both the Cre vector sequence and the LoxP vector sequence as it is.

Here, Non-Patent Document 1 discloses a technology that utilizes a CREM (Cre-M) sequence in which an intron sequence of the human β-globin gene is inserted into the sequence of the Cre gene. This technology prevents the expression of the recombinase in E. coli by inserting an intron sequence into the recombinase gene. With this technology, it is believed that it is also possible to amplify in E. coli a Cre-LoxP vector that contains both the sequence of the Cre vector and the sequence of the LoxP vector, without expressing Cre in E. coli.

However, which intron sequence is inserted depends on the organism or cell type that is the subject of conditional gene expression analysis, so it is not necessarily versatile. The CREM technology of Non-Patent Document 1 uses introns from human genes, so there are limitations on the organisms that can be targeted. In addition, with this technology, the expression of a functional recombinase when introduced into a target organism depends not only on the activity of the promoter but also on the splicing efficiency of mRNA, making it difficult to predict the expression level in the cells to be analyzed.
Furthermore, there have been almost no reports of analyses at the individual organism level using the CREM technique.

The inventors have been conducting extensive research into conditions under which spontaneous recombination does not occur in growing cells such as E. coli without inserting an intron into the Cre gene.

The present invention has been made in consideration of the above circumstances, and aims to provide a nucleic acid for gene expression that can be used in a highly versatile gene expression vector that can stably amplify a conditional gene expression vector, can be used for gene expression analysis in a wide range of organisms and cell types, can predict the expression level, and can be used for analysis at the individual organism level; a gene expression vector using the same; a method for producing the same; and a gene expression method.

Embodiments of the present invention have the following aspects.
Aspect 1 of the present invention is a nucleic acid used in a gene expression vector containing a recombinant enzyme expression gene, the nucleic acid for gene expression containing a sequence in which TA is repeated five or more times, to be introduced upstream of the recombinant enzyme expression gene.

Aspect 2 of the present invention is a nucleic acid for gene expression according to aspect 1, which contains a sequence in which the TA is repeated 5 to 23 times.

Aspect 3 of the present invention is a nucleic acid for gene expression according to aspect 1 or 2, which contains a sequence in which the TA is repeated 9 times.

Aspect 4 of the present invention is a nucleic acid for use in a gene expression vector containing a recombinant enzyme expression gene, the nucleic acid for gene expression containing a sequence having 50% or more homology with the SOSBox sequence (TACTGTATATATATACAGTA) for introduction upstream of the recombinant enzyme expression gene.

Aspect 5 of the present invention is a nucleic acid for use in a gene expression vector containing a recombinant enzyme expression gene, the nucleic acid for gene expression containing a sequence that has homology to the sequence of the -1700 to -900 site of the CarA promoter, and is to be introduced upstream of the recombinant enzyme expression gene.

Aspect 6 of the present invention is a gene expression vector comprising a nucleic acid for gene expression according to any one of aspects 1 to 5, a recombinant enzyme expression gene, a substrate sequence that serves as a substrate for the recombinant enzyme expression gene, and a planned recombination sequence sandwiched between the substrate sequences.

Aspect 7 of the present invention is the gene expression vector according to aspect 6, in which the recombinase expression gene contains a Cre gene sequence, the gene expression nucleic acid is contained upstream of the recombinase expression gene, and the substrate sequence contains a LoxP sequence.

Aspect 8 of the present invention is the gene expression vector according to aspect 6 or 7, in which the gene expression vector is used for gene expression using the Cre-LoxP system.

Aspect 9 of the present invention is a gene expression vector according to aspect 7 or 8, in which an insulator sequence is inserted between the sequence of the recombinase expression gene and the intended recombination sequence.

Aspect 10 of the present invention is a gene expression vector according to any one of aspects 7 to 9, which is configured so that an insulator sequence is inserted into the binding site between the genome sequence of the organism into which the gene expression vector is introduced and the gene expression vector.

Aspect 11 of the present invention is a method for producing a gene expression vector, which comprises introducing a nucleic acid for gene expression according to any one of aspects 1 to 5 into a nucleic acid sequence of a gene expression vector.

Aspect 12 of the present invention is the method for producing a gene expression vector according to aspect 11, further comprising a step of propagating the gene expression vector using E. coli, the E. coli being a Stbl strain or a derivative thereof, and being cultured at 30°C or lower.

Aspect 13 of the present invention is a method for gene expression, which comprises introducing into an organism a gene expression vector containing the nucleic acid for gene expression according to any one of aspects 1 to 6.

Aspect 14 of the present invention is the gene expression method according to aspect 13, in which the organism is a mouse, an African clawed frog, or a Japanese fire belly newt.

The present invention provides a nucleic acid for gene expression that can be used for a highly versatile gene expression vector that can stably amplify a conditional gene expression vector, can be used for gene expression analysis in a wide range of organisms and cell types, can predict the expression level, and can be used for analysis at the individual organism level, as well as a gene expression vector using the same, a method for producing the same, and a gene expression method.

FIG. 1 is a schematic diagram showing sequences used in searching for sequence elements that inhibit spontaneous recombination in Test Example 1. FIG. 1 is a schematic diagram showing sequences used in examining conditions for inhibiting spontaneous recombination in Test Example 2. FIG. 1 is a photograph showing the extraction of nucleic acid from a LoxP region in Test Example 2. FIG. 1 is a photograph showing extraction of nucleic acid from the CarA-CreER ^T2 site in Test Example 2. This is a photograph showing the extraction of full-length Transgene Target nucleic acid in Test Example 2. This is a graph showing the amount of nucleic acid obtained in the Stbl3 E. coli strain and the NEB Stable E. coli strain in Test Example 2. FIG. 1 is a schematic diagram showing sequences used in examining sequences that suppress spontaneous recombination in Test Example 3. FIG. 13 is a photograph showing the extraction of nucleic acid from the LoxP region in Test Example 3. FIG. 13 is a photograph showing extraction of nucleic acid from the PRE65-CreER ^T2 site in Test Example 3. This is a photograph showing the extraction of full-length Transgene Target nucleic acid in Test Example 3. FIG. 1 is a schematic diagram of plasmid DNA used in Test Example 4. FIG. 1 is a schematic diagram of the design of the KI construct in Test Example 4. FIG. 13 is a photograph showing fluorescent observation of the tail of a knock-in mouse in Test Example 4. FIG. 13 is a photograph showing the results of PCR analysis of genomic DNA of knock-in mice in Test Example 4. FIG. 13 is a photograph showing fluorescent observation of stages 10 and 43 of transgenic red-bellied newts in Test Example 5. FIG. 13 is a photograph showing fluorescent observation of stage 59 of a transgenic red-bellied newt in Test Example 5. FIG. 13 is a photograph showing the results of immunostaining of frozen sections of the eyeball of a transgenic red-bellied newt in Test Example 5. FIG. 2 is a schematic diagram of an experiment in Test Example 6 of this embodiment. FIG. 1 is a photograph showing fluorescence in skeletal muscles of the limb before and after tamoxifen administration.

The following describes the nucleic acid for gene expression, the gene expression vector using the nucleic acid, the method for producing the same, and the gene expression method according to the present invention, with reference to the embodiments. However, the present invention is not limited to the following embodiments.

(Nucleic acid for gene expression)
The nucleic acid for gene expression of this embodiment is a nucleic acid used in a gene expression vector containing a recombinant enzyme expression gene, and contains a sequence in which TA is repeated five or more times in order to be introduced upstream of the recombinant enzyme expression gene.

Nucleic acid for gene expression broadly refers to nucleic acid used in manipulations related to the expression of genes in an organism. Such nucleic acid can be either DNA or RNA. Manipulations related to gene expression broadly refer to manipulations using DNA or RNA, but in this embodiment, it refers to a nucleic acid sequence used to incorporate a specific sequence into a gene expression vector. For example, it is a DNA sequence used to be inserted into a gene expression vector.

The nucleic acid for gene expression is a nucleic acid for use in a gene expression vector containing a recombinase expression gene, and is introduced upstream of the recombinase expression gene. Here, the recombinase expression gene is a gene that causes recombination of a specific sequence by a recombinase expressed by the recombinase expression gene. Preferably, the recombinase is selected so as to exhibit the function of recognizing a specific substrate sequence and causing recombination of a planned recombination sequence, which is the specific sequence. Specific examples of the recombinase expression gene, substrate sequence, planned recombination sequence, and gene expression vector will be described later.

The nucleic acid for gene expression contains, for example, a sequence that repeats the TA (thymine-adenine) sequence five or more times. As a guideline, a sequence that repeats TA 5 to 23 times can be used. 5 or more TAs are sufficient, but the length can be appropriately selected depending on the conditions for amplifying the nucleic acid, convenience for other operations, etc.

Furthermore, TA is preferably repeated 8 to 10 times, and more preferably repeated 9 times (SEQ ID NO: 1). In this specification, a sequence in which TA is repeated 9 times may be referred to as a (TA)9 sequence, a TAx9 sequence, a TAX9 or a 9xTA sequence, etc. Furthermore, a sequence in which the (TA)9 sequence is repeated twice (i.e., 18 times of TA) may be referred to as a (TA)9X2 sequence.
With 9 repetitions of TA, the effect of this embodiment of suppressing spontaneous recombination can be fully exerted, and the sequence is unlikely to be too long, causing secondary structures or interactions that would reduce the effect. Although it depends on the conditions, a sequence with 9 repetitions of TA can exert a particularly strong effect of suppressing spontaneous recombination compared to other sequences.
SEQ ID NO:1: TATATATATATATATA

In another embodiment of the nucleic acid for gene expression, the nucleic acid comprises a sequence having 50% or more homology with the SOSBox sequence (SEQ ID NO: 2).
SEQ ID NO: 2: TACTGTATATATATACAGTA

The SOSBox sequence is known as a LexA repressor binding motif in E. coli. Its function is known to be related to the SOS response, which activates RecA and dissociates LexA when DNA damage occurs. The SOS response is involved in the arrest of cell division and DNA repair involving mutations, and is thought to contribute to the acquisition of antibiotic resistance.

The present inventors found that when a vector for gene expression using the Cre-LoxP system was prepared by inserting a CarA promoter derived from the Xenopus genome upstream of a Cre recombinase sequence, spontaneous recombination and removal of the site flanked by the LoxP sequences did not occur in E. coli. Thus, a search was conducted for a gene that would function to prevent spontaneous recombination, and the search was narrowed down to the sequence of SEQ ID NO:3.
SEQ ID NO: 3: ACAATATGGATTAATATATATATATATATATATATATATATATGTGCACCCGAGCCGAACCCACCCTTCCTCCACCCTTTTATAGACCCGCGCCCAGGCACTCAGCTATAAAAGCGAAAGTCGGAT

It was believed that the sequence of SEQ ID NO:3 or a sequence having partial homology to a part of it would function to prevent spontaneous recombination. SEQ ID NO:3 was similar to the SOSBox sequence. Furthermore, the researchers focused on a sequence of consecutive TAs in the SOSBox and found that this sequence functions to prevent spontaneous recombination, thus completing the present invention.

Furthermore, it is more preferable that the sequence having homology to the SOSBox sequence contains a portion of the SOSBox sequence in which five consecutive TAs occur.

It is also preferred that the nucleic acid for gene expression contains a sequence having homology to the sequence at positions −1700 to −900 of the CarA promoter.
The CarA (Cardiac Actin) promoter includes the full-length sequence of the CarA (Xl_CarA) promoter in the Xenopus genome from -3167 to +133 (Xenopus laevis strain J_2021 chromosome 8L, Xenopus laevis v10.1, NC_054385). Xl_CarA has an AT-rich region at -1700 to -900.
The sequence of the −1700 to −900 region of the CarA promoter analyzed by the present inventors is shown in SEQ ID NO:4.
SEQ ID NO: 4: CTTTCTGTTATTTCTATAGACAAATGACATTTGCATTAAAATATATATATATATATATATATATATATATATATATATATATATATATATATATAGTCAAAGAGGGACTAGGTTTCCAAAACTCAGCAATTACACCGTGATATTACACAAATTACTGAGTTTTAAACGAGTGCTGGTCCTCTTTACAATTATTTATATACTATTTTGACCAAGCACCCGGAAAACAGCATATTGTGAAGGAGTGCGGCTATCATCTGCATTATTGTTATATATATATATATAGTCCATAGAGATGC
CAGCACTCTTGAAAAAATGTAGTTACTGCCTGGGTGCAGAGCCACAGCAGCATTATTCAATTCGAAAAGAAGTCCAATCATTCAGGTCTTAAAGAACAAAAATATATTTAAGTTTATTACTGGGAGCGTAATGTTTCGGGCCAGCAATCTGGCCTTCCTCAAAGTTAGTCTCTCAGTAATAAACGTAAATATATTTTTTGTTCTTTACGACCTGAGTCTGCGAACTTCTTTTCGAATTATATATATATGCATTAGTGATGTGCGGGCTGGCCCGATACCCTCAGTACC
CGCAGGTCGGGTGGGTTCAGGCTGACCTCGCACCCATATTTGCGGGTGGTGGACCACCTTACACTGCTGGCATCCGACTTTCGCTTTTATAGCTGAGTGCCTGGGCGCGGGTCTATAAAAGGGTGGAGGAAGGGTGGGTTCGGCTCGGGTGCACATTATATATATATATATATATTAATCCATATTGTTGGCAAAACAAAC

By using a nucleic acid containing a sequence having homology to the sequence of the -1700 to -900 site of the CarA promoter as the nucleic acid for gene expression, spontaneous recombination can be prevented.
The present inventors have found that spontaneous recombination can be prevented by placing the Xl_CarA promoter upstream of the Cre sequence, and that spontaneous recombination can also be prevented by using the -1700 to -900 site of the Xl_CarA promoter, which has an AT-rich region.

The nucleic acid for gene expression may contain a single sequence or multiple sequences. For example, it may contain a sequence of 5 or more consecutive TAs, or a sequence having homology to the SOSBox sequence may be consecutive 2 or more times. It may also be consecutive via a sequence other than TA.

(Gene Expression Vector)
The gene expression vector of this embodiment comprises any of the above-described nucleic acids for gene expression, a recombinase expression gene, a substrate sequence that serves as a substrate for the recombinase expression gene, and a planned recombination sequence sandwiched between the substrate sequences.

As a gene expression vector, various vectors that are conventionally used for gene expression manipulation can be used. In this embodiment, for example, a plasmid vector can be used.

The combination of the recombinase expression gene, the substrate sequence, and the planned recombination sequence is selected so that the recombinase expressed by the recombinase expression gene exhibits the function of recognizing the substrate sequence and causing recombination with the planned recombination sequence. In this embodiment, the Cre-LoxP system described above is used. That is, the recombinase expression gene is a Cre gene that expresses the Cre recombinase (E. coli targeting P1) enzyme, the substrate sequence is LoxP, and the planned recombination sequence is sandwiched between the LoxP sequences. In this embodiment, the gene expression vector preferably contains a Cre gene sequence for the recombinase expression gene, the gene expression nucleic acid upstream of the recombinase expression gene, and the substrate sequence contains a LoxP sequence. The gene expression vector may be configured, for example, from upstream to downstream, with a gene expression nucleic acid, a recombinase expression gene, a substrate sequence, and a planned recombination sequence sandwiched between the sequences.

By using the gene expression nucleic acid of this embodiment, spontaneous recombination can be prevented, so that when the nucleic acid of the vector sequence having the above sequence is propagated using E. coli or the like, the planned recombination sequence is not removed by spontaneous recombination. Therefore, a gene expression vector having a Cre gene, a LoxP sequence, and a planned recombination sequence sandwiched between the LoxP sequences in one vector sequence can be produced. Recombination of the planned recombination sequence can be carried out by introducing this gene expression vector into an organism in which it is desired to carry out recombination under certain conditions, and there is no need to crossbreed the organisms or pass through generations.

The gene expression vector is preferably one used for gene expression using the Cre-LoxP system.
The Cre-LoxP system is a gene recombination system that uses a mechanism in which a target recombination sequence sandwiched between LoxP sequences of a substrate sequence is removed by the action of a recombinase, Cre. Specifically, a Cre vector having a Cre gene that is a recombinase expression gene, a LoxP sequence, and a LoxP vector having a target recombination sequence sandwiched between the LoxP sequences are introduced into an organism.

The gene expression vector used for gene expression using the Cre-LoxP system of this embodiment preferably has a Cre gene, a LoxP sequence, and a planned recombination sequence sandwiched between the LoxP sequences in one vector sequence, as described below.

Furthermore, the gene expression vector of this embodiment can be applied to site-specific gene recombination techniques, not limited to the Cre-LoxP system. Examples of site-specific gene recombination techniques include the FLP-FRP system.

In the gene expression vector of this embodiment, it is preferable that an insulator sequence is inserted between the sequence of the recombinase expression gene and the intended recombination sequence.
Here, the insulator sequence is a factor that regulates the expression of genes spaced apart in the sequence, and is believed to mainly inhibit transcription enhancers in eukaryotes and block chromatin changes, etc., and has been reported for various biological species. The insulator sequence can be appropriately selected depending on the biological species in which the gene expression vector is used and other conditions, and multiple types or multiple sequences may be used.
In this embodiment, the insulator sequence is the HS4 core insulator described in the Examples. In this embodiment, 2×HS4, in which two HS4s are linked in tandem, is inserted between a site containing a Cre gene sequence and a site containing LoxP.

The gene expression vector of this embodiment has an insulator sequence inserted between the sequence of the recombinase expression gene and the intended recombination sequence, which makes it possible to suppress interactions between the respective sequence sites (cassettes). This makes it possible to particularly enhance the effect of the present invention in preventing spontaneous recombination.

The gene expression vector of this embodiment is preferably configured so that an insulator sequence is inserted into the binding site between the genome sequence of the organism into which the gene expression vector is introduced and the gene expression vector.

For example, by providing an insulator sequence at both ends of the introduction construct, which is the sequence to be introduced into the genome, in the gene introduction vector, the insulator sequence can be inserted into the binding site between the genome sequence and the gene expression vector. When a restriction enzyme or the like is used for gene introduction, the insulator sequence can be provided on the introduction construct side just inside the recognition sequence of the restriction enzyme.

Other components of the gene expression vector may be those known in the art. For example, when a Cre gene sequence is used as the recombinase expression gene and a LoxP sequence is used as the substrate sequence, a promoter for expression under certain conditions may be provided upstream of the Cre gene sequence. The promoter may be, for example, a promoter for eukaryotic cells, or a promoter that is expressed in certain cells or tissues.
For example, a tissue-specific promoter may be arranged as a promoter for expression under certain conditions. By providing a tissue-specific promoter, it is possible to set conditions for Cre-induced recombinase expression in a specific tissue. In addition, a universal promoter without specific expression conditions may be arranged upstream of the LoxP sequence, or a promoter for expression under certain conditions may be arranged. By arranging a universal promoter, recombination is carried out depending on the expression conditions of the promoter arranged on the Cre side.
Here, "upstream" means the 5' side, but when a LoxP sequence or the like is arranged in the reverse direction, the 3' side may be upstream.
The nucleic acid for gene expression may be provided, for example, further upstream of the promoter.

As a specific example of how to prepare a gene expression vector, for example, in order to control the expression of a recombinant enzyme, a gene expression vector in which a known promoter sequence is combined with the sequence of this embodiment can be used and introduced.
For example, by combining this with the CRISPR/Cas9 knock-in technique into ROSA26, it can be used in a method for producing conditional gene expression mice, such as Cre-LoxP mice, in a single generation.
Furthermore, for example, in order to enhance cell/tissue specificity, a vector can be prepared in combination with split cre technology utilizing two different promoters.

(Method for producing gene expression vector)
In the method for producing a gene expression vector of this embodiment, the nucleic acid for gene expression is introduced into a nucleic acid sequence of a gene expression vector.
The production of nucleic acid for gene expression and its introduction into a gene expression vector can be carried out using conventional nucleic acid editing techniques.

The method for producing a gene expression vector of this embodiment further includes a step of propagating the gene expression vector using E. coli. Conventionally known methods can be used for propagating the gene expression vector in E. coli and the specific operations thereof.

As the E. coli, it is preferable to use an E. coli strain that is suitable for amplifying unstable DNA containing a repetitive sequence. As such an E. coli strain, it is preferable to use the Stbl strain or a derivative thereof. As the Stbl strain, it is preferable to use, for example, the Stbl3 strain. The E. coli strain is less susceptible to random recombination, and spontaneous recombination can be further prevented.

In the step of propagating the gene expression vector in E. coli, the culture is preferably performed at 30° C. or lower. Also, the culture is more preferably performed at 28° C. Spontaneous recombination can be further prevented by culturing at a temperature lower than the temperature at which E. coli is usually cultured. The reason for this is thought to be that excessive enzyme activity can be prevented.
More preferably, this step is carried out using the Stbl strain or a derivative thereof and culturing at 30° C. or lower.

(Gene Expression Method)
The gene expression method of this embodiment includes a method of introducing a gene expression vector into an organism, wherein the gene expression vector includes the nucleic acid for gene expression.

Gene expression vectors can be introduced into cultured cells for use. Gene expression vectors can also be directly introduced into living organisms for use. To introduce the vector into an organism, for example, it can be introduced into a fertilized egg.

Any organism may express the gene, but for example, mice, African clawed frogs, or red-bellied newts can be used. Mice and African clawed frogs can effectively express genes in this embodiment. Red-bellied newts take a long time, more than a year and a half, to change generations, so this embodiment is extremely effective in that the Cre-LoxP system can be used in individuals of the generation into which the gene has been introduced.

As a method for use in mice, for example, Cre-LoxP mice can be produced in one generation by combining with the above-mentioned CRISPR/Cas9-based knock-in technique into ROSA26.
Furthermore, when an endogenous enhancer/promoter is used, this technology can be used to generate TRE3G>Cre-LoxP mice in one generation and cross them with separately generated rtTA mice to generate Tet mice in a short period of time.

(Effects of this embodiment)
According to the present embodiment, there are provided a nucleic acid for gene expression that can be used for a highly versatile gene expression vector that can stably amplify a conditional gene expression vector, can be used for gene expression analysis in a wide range of organisms and cell types, can predict the expression level, and can be used for analysis at the individual organism level, a gene expression vector using the same, a production method thereof, and a gene expression method.

This embodiment enables the amplification of an integrated conditional gene expression vector in E. coli, similar to the conventional CREM technology that inserts an intron into a CRE sequence, but unlike the CREM technology, it is highly versatile and allows conditional gene expression analysis of a wide range of organisms and cell types. In addition, since the expression of the recombinant enzyme depends only on the activity of the promoter, it is easy to predict the expression level in the cells to be analyzed. Furthermore, since it can be applied without limiting organisms or cell types, it can also be easily applied to the analysis of organisms at the individual level.

This embodiment can significantly reduce the time and cost required for conditional gene expression in life science research. It is particularly effective in research using individual organisms.

According to this embodiment, genetically modified individuals can be produced in a short time and at low cost for various animals used in life sciences, such as rodents such as mice, and large animals such as pigs and monkeys, and therefore can also be used in industries such as contract production.
Furthermore, this embodiment can also be applied to the development of various vectors and kits used for the artificial expression and functional analysis of genes and proteins.

The following are examples. Note that the present invention is not limited to these examples.

(Test Example 1: Narrowing down of spontaneous recombination-inhibiting elements)
The present inventors have found that when a vector gene sequence has a Cre sequence and an enzyme expression gene sandwiched between LoxP sequences, if the CarA (cardiac actin) promoter derived from the Xenopus laevis genome is present upstream of the Cre sequence, the enzyme activity of spontaneous recombination of Cre is not expressed in Escherichia coli, and the sequence sandwiched between the LoxP sequences is preserved.
Therefore, the inventors investigated which sequence elements in the CarA sequence inhibit spontaneous recombination.

The sequences used to search for sequence elements that inhibit spontaneous recombination are shown in Figure 1. In one sequence, a vector gene was designed that had a partial sequence of the Xenopus CarA promoter (Xl_CarA promoter), a Cre sequence having Cre recombinase, a ubiquitous promoter and a dsRed gene sandwiched between LoxP, and a LoxP sequence having EGFP.
This vector gene was prepared by conventional nucleic acid editing technology and introduced into E. coli (Stbl3 strain) by heat treatment. E. coli was thinly spread on an agarose plate and cultured at 30° C. for 20 hours or more. The E. coli colonies that appeared were collected and grown in liquid medium, and the vector gene nucleic acid was extracted and examined for the presence or absence of the dsRed gene by PCR.
When the dsRed gene is detected among the partial sequences of the Xl_CarA promoter introduced, the dsRed gene flanked by LoxP is preserved, and the partial sequence is a sequence element that prevents spontaneous recombination.

As a result, it was revealed that the sequence of SEQ ID NO: 3, which is adjacent to the 3' end of the basal promoter of the Xl_CarA promoter, is a sequence element that prevents spontaneous recombination.
As a result of searching for a motif similar to the sequence of SEQ ID NO: 3, it was found that this sequence has similar motifs in the genomes of various organisms, and was similar to the LexA repressor binding motif (SOS box) of Escherichia coli (SEQ ID NO: 2 above). This sequence has an AT-rich region, particularly a region with eight consecutive TAs.

The CarA sequence has an AT-rich region at -1700 to -900 of the full-length sequence -3167 to +133 (Xenopus laevis strain J_2021 chromosome 8L, Xenopus laevis v10.1, NC_054385). This region closely matches the LexA repressor binding motif (SOS box).

(Test Example 2: Examination of conditions for inhibiting spontaneous recombination)
Before further examination of conditions, conditions for inhibiting spontaneous recombination were examined using the full-length CarA promoter.
The 12 kb Transgene Target shown in Figure 2 was used. This sequence has the CreER ^T2 sequence of the recombinase expression gene downstream of the CarA promoter (CarA-CreER ^T2 site, 6.1 kb), and upstream of it, in the reverse orientation, the sequences of EGFP and SpA, which are the intended recombination sequences, sandwiched between the substrate sequences LoxP (LoxP region).
The LoxP region is 2.5 kb long if spontaneous recombination does not occur, and 1.5 kbp long if spontaneous recombination occurs.
Between the sequence having Cre and the sequence sandwiched between LoxP and on both ends of the sequence, 2×HS4 insulator sequences in which two HS4 core insulators are linked in tandem are arranged.
The HS4 core insulator was developed by Dr. Gary Felsenfeld of the National Institutes of Health (NIH), Bethesda, MD, USA, and is used with the kind permission of our university, the MTA of the NIH, and Dr. Gary Felsenfeld.

A vector gene containing this 12 kb Transgene Target was created using conventional nucleic acid editing technology and introduced into E. coli (Stbl3 strain, NEB Stable strain) by heat treatment. E. coli was thinly seeded onto an agarose plate and cultured at 30°C for more than 20 hours. The E. coli colonies that appeared were collected and grown in liquid medium, the vector gene nucleic acid was extracted, and the nucleic acid of the desired site/region was obtained by enzyme treatment, and the length and amount of the grown nucleic acid were examined by agarose gel electrophoresis.

Figure 3 shows the nucleic acid extracted from the LoxP region. As shown in the figure, a band at 2.5 kb, i.e., the nucleic acid from the LoxP region where spontaneous recombination was prevented, was confirmed in both the Stbl3 E. coli strain and the NEB Stable E. coli strain.

4 shows the extraction of nucleic acid from the CarA-CreER ^T2 site. A band at the position of 6.1 kb, i.e., the nucleic acid from the CarA-CreER ^T2 site, was confirmed in both the Stbl3 E. coli strain and the NEB Stable E. coli strain.

Figure 5 shows the extraction of the full-length 12 kb Transgene Target nucleic acid. The 12 kb Transgene Target is cleaved at both ends by the I-Scel enzyme. A band at the 12 kb position, i.e., the nucleic acid of the 12 kb Transgene Target, was confirmed in both the Stbl3 E. coli strain and the NEB Stable E. coli strain.

Figure 6 shows the amount of nucleic acid obtained from the Stbl3 E. coli strain and the NEB Stable E. coli strain. The amount of nucleic acid was measured using a microspectrophotometer. Both E. coli strains were able to grow and recover sufficient amounts of nucleic acid.

The results of these figures show that by placing the CarA promoter upstream, it is possible to amplify nucleic acids having a Cre gene (recombinase expression gene) and a LoxP region (substrate sequence and planned recombination sequence) on one sequence, and that proliferation is possible while suppressing spontaneous recombination in both the Stbl3 E. coli strain and the NEB Stable E. coli strain.

(Test Example 3: Examination of spontaneous recombination inhibitor elements)
Further investigation was carried out to determine which motifs in the CarA promoter and SOSBox sequence are capable of suppressing spontaneous recombination, and the essential sequences and their effects.
The 9.7 kb Transgene Target shown in Figure 7 was used. This sequence has a Cre sequence (cpRPE65-CreER ^T2 site, 3.3 kb) having a pigment cell promoter cpRPE65 and a recombinase expression gene CreER ^T2 sequence, and upstream of it in the reverse orientation, the sequences of EGFP and SpA, which are intended recombination sequences, sandwiched between LoxP substrate sequences (LoxP region).
The LoxP region is 2.5 kb long if spontaneous recombination does not occur and 1.5 kbp if spontaneous recombination occurs, and the total length of this sequence of 9.7 kb becomes 8.7 kb if recombination occurs.
Between the sequence having Cre and the sequence sandwiched between LoxP and on both ends of the sequence, 2×HS4 insulator sequences in which two HS4 core insulators are linked in tandem are arranged.
An SOS RE sequence is located upstream (3' side) of cpRPE65. This sequence has partial homology to the SOS box, but uses a sequence of nine TA sequences ((TA)9 sequence), a sequence of nine TA sequences repeated twice ((TA)9X2 sequence), or a sequence of nine TA sequences repeated twice with GGG in between ((TA)9X2GGG sequence).

A vector gene containing this 9.7 kb Transgene Target was created using conventional nucleic acid editing technology and introduced into E. coli (Stbl3 strain, NEB Stable strain) by heat treatment. E. coli was thinly spread on an agarose plate and cultured at 30°C for more than 20 hours. The E. coli colonies that appeared were collected and grown in liquid medium, the vector gene nucleic acid was extracted, and the nucleic acid of the desired site/region was obtained by enzyme treatment, and the length and amount of the grown nucleic acid were examined by agarose gel electrophoresis.

Figure 8 shows the nucleic acid extracted from the LoxP region. As shown in the figure, a band at 2.5 kb was confirmed for the (TA)9 sequence in the Stbl3 E. coli strain, and for the (TA)9X2 sequence and (TA)9X2GGG sequence in the NEB Stable E. coli strain, that is, the nucleic acid of the LoxP region where spontaneous recombination was prevented.

9 shows the extraction of nucleic acid from the PRE65-CreER ^T2 site. As shown in the figure, a band at 3.3 kb, i.e., nucleic acid from the PRE65-CreER ^T2 site, was confirmed for the (TA)9 sequence in the Stbl3 E. coli strain, and for the (TA)9X2 sequence and (TA)9X2GGG sequence in the NEB Stable E. coli strain.

Figure 10 shows the extraction of the full-length 9kb Transgene Target nucleic acid. The 9kb Transgene Target is cleaved at both ends by the I-Scel enzyme. As shown in the figure, the (TA)9 sequence was confirmed in the Stbl3 E. coli strain, and the (TA)9X2 sequence and (TA)9X2GGG sequence were confirmed in the NEB Stable E. coli strain, meaning that the nucleic acid of the 9kb Transgene Target was confirmed.

The above results show that spontaneous recombination can be prevented by any of the following sequences that share partial homology with the SOS box: a sequence with nine TA sequences ((TA)9 sequence, TAX9 sequence), a sequence with nine TA sequences repeated twice ((TA)9X2 sequence), or a sequence with nine TA sequences repeated twice with GGG in between ((TA)9X2GGG sequence).
In particular, even a sequence with nine TA sequences (a total of 18 base pairs) was able to grow in the Stbl3 strain while preventing spontaneous recombination. The reason why spontaneous recombination was prevented even with a short nucleic acid sequence is thought to be that nonspecific enzyme reactions were suppressed in the Stbl3 strain, effectively preventing spontaneous recombination.

(Test Example 4: Preparation of CreER ^T2 -LoxP-introduced mice)
Mice were generated in which the CreER ^T2 -LoxP vector was knocked into the ROSA26 region of chromosome 6 by CRISPR-Cas9 homologous recombination.
Fig. 11 is a schematic diagram of the plasmid DNA used in this test example. The configuration of the transfer cassette is basically the same as that in Test Example 3 (Fig. 7), but cpRPE65 was changed to HACTA1, a human skeletal muscle-specific promoter.

The sequence information for HACTA1 (Acta1_human_promoter_-2000_to_+239_ Homo sapiens actin alpha 1, skeletal muscle (ACTA1), RefSeqGene (LRG_429) on chromosome 1) is shown in SEQ ID NO:5.

SEQ ID NO: 5: AAGGCCCAAATGTAAGCTAGTCCCCTTACGTTACATGCAGCTCATTTGCTAAGTGGTTTTTTTCTAGTATCTCCACTACTCGCTGACACAGGAGGACACAGGATGTTAAAAAGGAAATACAGTTCTGTCAATTATTCACTTACTCTCCAAAATACTTGGAAGAACTAAATATGGAACCATAGGAGACTTTATCCTCACCGCATAGTCCCTATACTAGTCAAACTCCTTATTTTTTAATTGATCATTTTTAGGAAGGTAGCATTTTATTCACTAGAACATTTTTGTTAA
TACTTGTTTATTTTTGGGATGAACTGCCATGATGTGGGCTACAGAGGAGGGTCGCATATGCTTCCATCCCCCTTTTAGAGAATCCACACCTGTCCCAGTTGCTGGGTTCCACTACCAAAGTGAATTGCAACTATTTTAGGAGCACTTAAGCACATCCGAAAAATGAGTGATTCTGTTCTGGCCCACACCACATCACTGATGTACCCCCTTAAAGCATGTCCCTGAGTTCATCACAGAAGACTGCTCCTCCTGTGCCCTCCACAAGGTTAGAACTGTCCTTGTCTTAG
GGAAAAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGACAGGCACCAACTGGGTAACCTCTGCTGACCCCCACTCTACTTTACCATAAGTAGCTCCAAATCCTTCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAACCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAGCCCGGCCTTGGCAGGCATCATCCTCTAAATATAAAGATGAGTTTGTTCAGCCTTTGCAGAA
GGAAAAACTGCCACCCATCCTAGAGTGCCGCGTCCTTGTCCCCCCACCCCCTCCAATTTATTGGGAGGAAGGACCAGCTAAGCCTCATCTAGGAAGAGCCCCTCACCCATCTCCACCTCCACTCCAGGTCTAGCCAGTCCTGGGTTGTGACCCTTGTCTTTCAGCCCCAGGAGAGGGACACACATAGTGCCACCAAAGAGGCTGGGGGAGGGCCTCAGCCCACCAAAACCTGGGGCCAGTGCGTCCTACAGGAGGGGAACCCTCACCCCTTCAATCCCTTTAGGAGAC
CCAAGGGCGCTGCGCGTCCCTGAGGCGGACAGCTCCGTGTGCTCAGGCTTTGCGCCTGACAGGCCTATCCCCGGGAGCCCCCGCGCCTCCTCCCCGGCGCTCCGCCCTCGCCTCCCCCCGCCAGTTGTCTATCCTGCGACAGCTGCGCGCCCTCCGGCCGCCGGTGGCCCTCTGTGCGGTGGGGGAAGGGGTCGACGTGGCTCAGCTTTTTGGATTCAGGGAGCTCGGGGGTGGGAAGAGAGAAATGGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTTCCTTC
CCTTCCTGAGACTCAGGAGTGACTGCTTCTCCAATCCTCCCAAGCCCACCACTCCACACGACTCCCTCTTCCCGGTAGTCGCAAGTGGGAGTTTGGGGATCTGAGCAAAGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTTCCCGAGCGCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTTTGGGACAGGTGCGGTTCCCGGAGCGCAGGCGCACACATGCACCCACCGGCGAACGCGGTGACCCTCGCCCCACCCCATCCCCTCC
GGCGGGCAACTGGGTCGGGTCAGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGTCAACGCAGGGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGCCCAACACCCAAATATGGCTCGAGAAGGGCAGCGACATTCCTGCGGGGTGGCGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGCGGCCACCGCAGCGGACAG
CGCCAAGTGAAGCCTCGCTTCCCCTCCGCGGCGACCAGGGCCCGAGCCGAGAGTAGCAGTTGTAGCTACCCGCCCAGGTAGGGCAGGAGTTGGGAGGGGACAGGGGGACAGGGCACTACCGAGGGGAACCTGAAGGACTCCGGGGCAGAACCCAGTCGGTTCACCTGGTCAGCCCCAGGCCTGCGCCCTGAGCGCTGTGCCTCGTCTCCGGAGCCACACGCGC

Furthermore, at both ends of the transfer cassette, DNA (L. Arm and R. Arm) corresponding to the knock-in site within the ROSA26 region was placed instead of the I-SceI recognition DNA, enabling homologous recombination with the genomic DNA.
FIG. 12 is a schematic diagram of the design of the KI construct.

This plasmid DNA was amplified in NEB Stable E. coli strain (30°C, 20 hours or more). The amplified plasmid DNA was then extracted, purified, and concentrated.

This plasmid DNA was injected into mouse fertilized eggs together with Cas9 protein and gRNA, and the fertilized eggs were then transplanted into the oviducts of pseudopregnant mice. A total of 621 fertilized eggs were transplanted over two trials, with 88 mice born (birth rate of approximately 14%) and 78 mice surviving (survival rate of approximately 89%). Knock-in was confirmed in 20 of these mice (knock-in rate was approximately 26% of surviving individuals and approximately 3% of transplanted fertilized eggs). The results of two mice (M1 and M1) are shown here as examples. The tip of the tail was taken from each mouse and its fluorescence was examined.

13 is a photograph showing the fluorescent observation of the tail of a knock-in mouse. (a) shows the bright field of the tail tip, (b) shows EGFP, and (c) shows mCherry fluorescent signals. In the F0 (same generation) mice, the left side of the figure is M1 and the right side is M2, neither of which was administered TAM (tamoxifen). The scale bar in the figure indicates 4.5 mm.
All tails showed EGFP fluorescence but no mCherry fluorescence, indicating that the DNA region flanked by LoxP was not lost during the preparation of the plasmid DNA or during mouse development, i.e., spontaneous recombination by Cre had not occurred.

Next, genomic DNA was extracted from each tail tip and genotyping was performed by PCR. That is, a region containing the DNA sequence encoding CreER ^T2 (735 bp), a region containing the DNA sequence encoding EGFP (607 bp), and a region containing both the 3'-end of the transfer cassette and the knock-in sequence of the ROSA26 region (3.3 kbp) were amplified by PCR.

14 is a photograph showing the results of PCR analysis of the genomic DNA of the knock-in mouse. In the figure, IL-2 (324 bp) is a gene present on another chromosome, and is shown as a positive control to show the quantity and quality of the genomic DNA.
As shown in the figure, all genomic DNAs contained DNA encoding Cre or EGFP, but the transfer cassette was integrated into the ROSA26 region only in the genomic DNA of mouse M2. In other words, it was proven that knock-in was successful in mouse M2. In mouse M1, the transfer cassette is considered to have been randomly integrated into a genomic region other than the ROSA26 region.

(Test Example 5: CreER ^T2 -LoxP-introduced newt production)
A newt (Cynops pyrrhogaster) was prepared in which the CreER ^T2 -LoxP vector was integrated into the genome by the I-SceI method.
The configuration of the transfer cassette used in this test example was the same as that in Test Example 3. CreER T2 is expressed under the control of the RPE65 promoter (-1049 to +49; Casco-Robles et al., Transgenic Research 24: ^463-473 , 2015; DOI: 10.1007/s11248-014-9857-1), and recombination occurs in mature retinal pigment epithelial cells in the eyeball in the presence of tamoxifen.

The plasmid DNA was amplified in NEB Stable E. coli strain (30° C., 20 hours or more), and then the plasmid DNA was extracted, purified, and concentrated.
This plasmid DNA was injected into fertilized eggs of the newt together with I-SceI protein (R06945; New England Biolabs Japan, Tokyo, Japan; dilution buffer: 10 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM DTT, pH 8.8 at 25°C) and the eggs were raised as is (Casco-Robles et al., Nature Protocols 6: 600-608, 2011; DOI: 10.1038/nprot.2011.334). A total of 390 fertilized eggs were injected with the mixture of plasmid DNA and I-SceI protein (2 nl, plasmid DNA 100 pg, I-SceI 0.002 units) in 13 trials.

Figure 15 is a photograph showing fluorescent observation of stages 10 and 43 of transgenic red-bellied newts. (a) shows bright field, (b) shows EGFP, and (c) shows mCherry fluorescent signals. The upper side of the figure is stage 10 (St. 10), and the lower side is stage 43 (St. 43), and in neither case was 4-hydroxytamoxifen (4-OHT) administered. The scale bars in the figures all indicate 2 mm.

As shown in the figure, EGFP fluorescence was confirmed in 38 embryos at stage 10 (late blastula) (gene transfer rate of approximately 9.7%). However, no mCherry fluorescence was confirmed. These individuals developed normally. During this time, EGFP fluorescence was observed throughout the body, but no mCherry fluorescence was observed. An embryo at stage 43 (forelimb stage 6a) is shown as a sample here. This indicates that the DNA region flanked by LoxP was not lost during the preparation of the plasmid DNA or the development of the newt, i.e., spontaneous recombination by Cre did not occur.

The larvae were then reared in water containing 4 μM 4-hydroxytamoxifen (4-OH Tam; (Z)-4-hydroxytamoxifen (H7904-5MG; Merck, Sigma-Aldrich, Tokyo, Japan) dissolved in DMSO) for 2 days to induce Cre-mediated recombination. The water containing 4-OH Tam was replaced with fresh water every other day.

16 is a photograph showing fluorescence observation of stage 59 of a transgenic newt. (a) shows a bright field, (b) shows EGFP, and (c) shows mCherry fluorescent signals. The upper part of the figure (scale bar in the figure is 8 mm) and the lower part shows a partially enlarged view of the upper part (scale bar in the figure is 2 mm). The image is shown 48 hours after administration of 4-hydroxytamoxifen (4-OH Tam, 4-OHT).
As shown in the upper figure, no mCherry fluorescence was observed externally.

Next, the stage 59 eyeball was enucleated and fixed in a fixative (a modified Zamboni's solution: 2% (w/v) paraformaldehyde and 0.2% (w/v) picric acid in PBS (pH 7.4); Chiba et al., Journal of Comparative Neurology 495: 391-407, 2006; DOI: 10.1002/cne.2088 0) for 6 hours at 4°C, and 20 μm-thick frozen sections were prepared, and the expression patterns of RPE65 and mCherry proteins were examined immunohistochemically using RPE65 antibody (1:500; MAB5428; EMD Millipore, CA 92590, USA) and RFP antibody (for mCherry; 1:200-500; 600-401-379; Rockland Immunochemicals, PA 19468, USA).

Figure 17 is a photograph showing the results of immunostaining of frozen sections of the eyeball of a transgenic red-bellied newt. (a) Bright field, (b) DAPI/RPE65, (c) DAPI/DsRed, and (d) RPE65/DsRed fluorescent signals. The upper, middle, and lower parts of the figure each show an enlarged portion of the upper figure. The image is taken 48 hours after administration of 4-hydroxytamoxifen (4-OH Tam, 4-OHT). The scale bar in the upper figure is 350 μm, and the lower figure is 100 μm.

As shown in the figure, it was demonstrated that retinal pigment epithelial cells expressed mCherry protein.
These results demonstrate that the (TA)9 sequence does not inhibit the activity of tissue-specific promoters in the newt, unlike in E. coli, and that conditional genetic recombination is possible in a single generation.

(Test Example 6: Induction of recombination using CreER ^T2 -LoxP-introduced mice)
An experiment was carried out using inducible Cre, CreER ^T2 , in which tamoxifen (TAM) was injected into the abdominal cavity of adult mice to activate inducible Cre and induce recombination in target cells.

Figure 18 is a schematic diagram of this experiment. An adult mouse in which the CreER ^T2 -LoxP vector was knocked into the ROSA26 region of chromosome 6 by CRISPR-Cas9 expressed EGFP throughout the body, similar to the tail in Test Example 4, and knock-in into the relevant tissues could be confirmed by EGFP. Tamoxifen was injected intraperitoneally into this mouse, and mCherry expression specific to skeletal muscle fibrocytes was confirmed.

First, two males and two females were randomly selected from the F0 individuals in which knock-in had been confirmed by PCR, and the fluorescence of their skeletal muscles was examined. EGFP fluorescence was observed in the skeletal muscles of all individuals. However, mCherry fluorescence was also observed in half of the males and half of the females (these results are not shown). In these individuals, it is likely that the transfer cassette was not only knocked out into the Rosa26 region, but also randomly inserted into other regions in the genome. In fact, the EGFP fluorescence of these skeletal muscles tended to be stronger than that of skeletal muscles that did not show mCherry fluorescence. The mechanism by which mCherry was spontaneously expressed was unclear.

To investigate whether it is possible to artificially induce recombination using tamoxifen, the researchers irradiated the soles of the feet of mice with excitation light, selected mice in which no mCheery fluorescence was observed in their internal muscles, and then injected tamoxifen into the abdominal cavity of these mice.
Tamoxifen (T5648-1G; Merck, Sigma-Aldrich, Tokyo, Japan) solution dissolved in corn oil (032-17016; Fujifilm-Wako, Osaka, Japan) to a concentration of 20 mg/ml was intraperitoneally injected into the mice for two consecutive days at a dose of 50-200 mg tamoxifen/kg body weight per day.

Figure 19 is a photograph showing the fluorescence of the skeletal muscle of the limb before and after tamoxifen administration. Column (a) in the left half of the figure shows the fluorescence of the skeletal muscle of the limb before tamoxifen administration. Column (b) in the right half of the figure shows the fluorescence of the skeletal muscle of the limb one week after the two administrations of tamoxifen. (a) and (b) in the figure are images of different individuals. The scale bar in the figure indicates 5 mm.

As shown in (a) of the figure, no mCherry fluorescence was observed in the skeletal muscle of the limb before tamoxifen administration, and the fluorescence intensity was at the level of autofluorescence.
As shown in (b) of the figure, mCherry fluorescence was observed in the skeletal muscle of the limb one week after tamoxifen administration.
These results indicate that recombination can be induced by tamoxifen. The reason that EGFP fluorescence was observed even after recombination is thought to be because recombination does not occur in all nuclei of multinucleated myofiber cells.

The success of artificial recombination induced by tamoxifen not only demonstrated that (TA)9 does not inhibit the activity of tissue-specific promoters in mice, as it does in E. coli, but also demonstrated that conditional genetic recombination is possible in a single generation.

In general, random insertion of DNA fragments derived from the introduced plasmid into the genome cannot be avoided in CRISPR-Cas9 KI. In this case, it is necessary to repeatedly cross with wild-type mice, remove individuals having chromosomes with randomly inserted DNA fragments by genotyping, and screen for pure KI mice. This technology has made it possible to produce Cre-LoxP mice in one generation, but this work is essential in order to use these mice for more accurate analysis.
With this technology, not only are the KI-processed genomic regions known, but there is also only one KI-processed construct, making it possible to carry out this task efficiently.

The present invention provides a nucleic acid for gene expression that can be used for a highly versatile gene expression vector that can stably amplify a conditional gene expression vector, can be used for gene expression analysis in a wide range of organisms and cell types, can predict the expression level, and can be used for analysis at the individual organism level, as well as a gene expression vector using the same, a method for producing the same, and a gene expression method.

Claims (14)

1. A nucleic acid used in a gene expression vector containing a recombinant enzyme expression gene,
   A nucleic acid for gene expression, which comprises a sequence repeating TA five or more times and is to be introduced upstream of the recombinant enzyme expression gene.
2. The nucleic acid for gene expression according to claim 1, which contains a sequence in which the TA is repeated 5 to 23 times.
3. The nucleic acid for gene expression according to claim 1, which contains a sequence in which the TA is repeated nine times.
4. A nucleic acid used in a gene expression vector containing a recombinant enzyme expression gene,
   A nucleic acid for gene expression, which comprises a sequence having 50% or more homology with an SOSBox sequence (TACTGTATATATATACAGTA), and which is to be introduced upstream of the recombinant enzyme expression gene.
5. A nucleic acid used in a gene expression vector containing a recombinant enzyme expression gene,
   A nucleic acid for gene expression, which comprises a sequence having homology to the sequence of the -1700 to -900 site of the CarA promoter, and is to be introduced upstream of the recombinant enzyme expression gene.
6. A nucleic acid for gene expression according to claim 1, 4 or 5,
   A recombinant enzyme expression gene;
   A substrate sequence serving as a substrate for the recombinant enzyme expression gene;
   A gene expression vector comprising a planned recombination sequence flanked by the substrate sequences.
7. The recombinase expression gene comprises a Cre gene sequence;
   The nucleic acid for gene expression is included upstream of the recombinant enzyme expression gene,
   The gene expression vector of claim 6 , wherein the substrate sequence comprises a LoxP sequence.
8. The gene expression vector according to claim 6, which is used for gene expression using the Cre-LoxP system.
9. The gene expression vector according to claim 6, in which an insulator sequence is inserted between the sequence of the recombinase expression gene and the intended recombination sequence.
10. The gene expression vector according to claim 6, which is configured so that an insulator sequence is inserted into the binding site between the genome sequence of the organism into which the gene expression vector is introduced and the gene expression vector.
11. A method for producing a gene expression vector, comprising introducing the nucleic acid for gene expression according to claim 1, 4 or 5 into a nucleic acid sequence of a gene expression vector.
12. The method further comprises a step of propagating the gene expression vector using E. coli,
    The method for producing a gene expression vector according to claim 11, wherein the Escherichia coli is a Stbl strain or a derivative thereof, and is cultured at 30°C or lower.
13. A method for gene expression, comprising introducing a gene expression vector containing the nucleic acid for gene expression according to claim 1, 4 or 5 into an organism.
14. The gene expression method according to claim 13, wherein the organism is a mouse, an African clawed frog, or a Japanese fire belly newt.

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