CN112239765A - Construction method of yeast fixed-point double-DSB synchronous induction model - Google Patents
Construction method of yeast fixed-point double-DSB synchronous induction model Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention relates to a construction method of a yeast site-specific double-DSB synchronous induction model, which is formed by integrating a GAL-I-SceI-KanMX transformation box on a saccharomyces cerevisiae genome through a chemical transformation method. The invention can synchronously induce the generation of two DSBs with different intervals by activating a promoter to express I-SceI endonuclease and identifying I-SceI recognition sites at two ends for cutting, thereby eliminating the interference of other damage types, more importantly, the model can be used for researching synchronously generated 'aggregated' DSBs which are characteristic damage types generated by cancer radiotherapy and a plurality of chemotherapeutic drugs, and the model is a powerful tool for researching cell signal pathways and drug toxicology. And the invention can synchronously generate a plurality of DSBs by slight improvement, and can be applied to cells of human or other organisms.
Description
Technical Field
The invention belongs to the genetic engineering technology, and particularly relates to a construction method of a yeast fixed-point double-DSB synchronous induction model.
Background
DSB is a very serious DNA damage that, if not repaired in time, can cause genetic mutations, induce cancer, and the like. DSBs are repaired mainly by Homologous Recombination (HR) and non-homologous end joining (NHEJ), and the construction of chemically or physically inducible DSBs is an essential tool for studying the DNA damage pathway described above. Traditional DNA damage models are induced by ionizing radiation, chemical mutagens and the like, cannot well limit damage types to single DSB damage, and are often accompanied by DNA Single Strand Breaks (SSB), base mutations, DNA double strand cross-linking and the like. In addition, the production of more than one DSB in cells under natural conditions is common, the toxicological effect when a plurality of DSBs are synchronously produced is researched, and the repair process of an organism has important biomedical basic theoretical significance and use value.
As shown in FIG. 1, DNA damage induced by external factors such as ionizing radiation and chemical mutagens is complex in type and random in site, and molecular signal pathways of specific repair DSBs such as HR and NHEJ cannot be effectively researched. A model capable of stably inducing and generating double DSBs in a fixed-point mode is constructed, the spacing distance between the two DSBs can be changed according to needs, and therefore interference of other damage types can be eliminated, and more importantly, special harm to organisms and a synergistic process of cell repair response of synchronously generated 'aggregated' DSBs relative to single DSBs can be researched. Since "aggregate" DNA damage is a characteristic type of damage produced by cancer radiotherapy and numerous chemotherapeutic drugs, this model is a powerful tool for studying cell signaling pathways and drug toxicology.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for constructing a fixed-point synchronous double-DSB model.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for constructing a yeast fixed-point double-DSB synchronous induction model comprises the following steps:
the method comprises the steps of designing an upstream primer and a downstream primer, adding I-SceI recognition sites at two ends of the primers, and amplifying an expression cassette of an I-SceI gene controlled by a saccharomyces cerevisiae inducible promoter and a KanMX screening gene GAL-I-SceI-KanMX from a plasmid pGSKU by using the upstream primer and the downstream primer;
designing a second section of primer for second PCR amplification, so that the homology arms of the upstream primer and the downstream primer are 70-80 bp;
thirdly, integrating the GAL-I-SceI gene sequence obtained by PCR amplification to a saccharomyces cerevisiae genome by using a conversion method, obtaining a successfully integrated strain by using a screening marker gene, and designing a verification primer to verify whether the construction is successful;
and a yeast containing GAL or other promoter controlled to express I-SceI gene, wherein when the promoter is activated, the I-SceI endonuclease can be expressed, and the I-SceI recognition sites placed at both ends in advance are recognized and cleaved, so that double DSB is generated.
In the first step, an upstream primer is SEQNO.1, and a downstream primer is SEQNO.2;
DNA polymerase reaction System: ddH2O15.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, DNA polymerase 0.5. mu.l, dNTP 1. mu.l at 2.5mM, 10 XPfuuffer 5. mu.l, DNA template 1. mu.l;
DNA polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
In addition, in the step, the upstream primer is SEQNO.3, and the downstream primer is SEQNO. 4;
DNA polymerase reaction System: ddH2O15.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, DNA polymerase 0.5. mu.l, dNTP 1. mu.l at 2.5mM, 10 XPfuuffer 5. mu.l, DNA template 1. mu.l;
DNA polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
And in the step three, the upstream primer is SEQNO.5, and the downstream primer is SEQNO.6.
In the first step, the saccharomyces cerevisiae inducible promoter is a galactose promoter GAL, a hexose transport vector or an alcohol dehydrogenase II.
In addition, the above-described procedure may introduce a segment of an unnecessary sequence by overlap extension PCR, if necessary, so as to adjust the distance between two DSBs.
In the step, the expression cassette of the I-SceI gene comprises a galactose promoter, an I-SceI endonuclease and a KanMX/hygromycin/nourseothricin screening marker.
The invention has the advantages and positive effects that:
1. the invention establishes a method for stably inducing and synchronously generating two DSBs at any site of a yeast genome, and can set the spacing distance of the two DSBs according to requirements. The method is slightly extended, namely a plurality of DSBs can be synchronously generated at any position of a genome, and the method is a powerful DNA repair response research model. Is particularly suitable for researching homologous recombination repair (HR) and non-homologous end joining repair (NHEJ) and a synergistic and complementary mechanism among different repair paths, and can be used for cells of human bodies or other organisms.
2. Compared with the traditional random DNA damage induced by ionizing radiation, chemical mutagens and the like, the double-DSB site-directed induction model established by the invention only induces the single DSB damage, can eliminate the interference of other types of DNA damage, and more importantly can induce the generation of the 'aggregated' DSB at any position of a genome, so that the special harm to an organism and the synergistic process of cell repair response when a plurality of DSBs are simultaneously generated can be accurately obtained, and the model is a strong and beneficial tool for researching the toxicity of anti-cancer drugs and accurate medical treatment.
3. The invention constructs a model which can stably induce and generate double DSBs at fixed points, and the spacing distance between the two DSBs can be changed according to needs, so that the interference of other damage types can be eliminated, and more importantly, the special harm to organisms and the synergistic process of cell repair response of synchronously generated 'aggregated' DSBs relative to a single DSB can be researched. Since "aggregate" DNA damage is a characteristic type of damage produced by cancer radiotherapy and numerous chemotherapeutic drugs, this model is a powerful tool for studying cell signaling pathways and drug toxicology.
Drawings
FIG. 1 is a schematic diagram of a prior art method for generating DSB damage;
FIG. 2 is a schematic diagram of a dual DSB synchronous induction model according to the present invention; DSBs of different spacer lengths can be generated by adjusting the length of the spacer sequence (DNA fragment with no genetic effect between genes);
FIG. 3 is a schematic diagram of the present invention for verifying success of a dual DSB fixed-point injury model; experimental groups 1-5 show that a 3620bp band is amplified, and the success of constructing a double DSB injury model with a 3620bp spacing sequence is shown.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
A method for constructing a yeast fixed-point double-DSB synchronous induction model comprises the following steps:
the method comprises the steps of designing an upstream primer and a downstream primer, adding I-SceI recognition sites at two ends of the primers, and amplifying an expression cassette of an I-SceI gene controlled by a saccharomyces cerevisiae inducible promoter and a KanMX screening gene GAL-I-SceI-KanMX from a plasmid pGSKU by using the upstream primer and the downstream primer;
designing a second section of primer for second PCR amplification, so that the homology arms of the upstream primer and the downstream primer are 70-80 bp;
thirdly, integrating the GAL-I-SceI gene sequence obtained by PCR amplification to a saccharomyces cerevisiae genome by using a conversion method, obtaining a successfully integrated strain by using a screening marker gene, and designing a verification primer to verify whether the construction is successful;
and a yeast containing a GAL or other promoter controlled expression of the SceI gene, wherein activation of the promoter allows expression of the I-SceI endonuclease, and further allows recognition and cleavage of the I-SceI recognition sites placed at both ends in advance, thereby generating the double DSB. As can be seen in fig. 2.
Preferably, an upstream primer in the step is SEQNO.1, and a downstream primer is SEQNO.2;
DNA polymerase reaction System: ddH2O15.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, DNA polymerase 0.5. mu.l, dNTP 1. mu.l at 2.5mM, 10 XPfuuffer 5. mu.l, DNA template 1. mu.l;
DNA polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
Preferably, the upstream primer is SEQNO.3, and the downstream primer is SEQNO. 4;
DNA polymerase reaction System: ddH2O15.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, DNA polymerase 0.5. mu.l, dNTP 1. mu.l at 2.5mM, 10 XPfuuffer 5. mu.l, DNA template 1. mu.l;
DNA polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
Preferably, the upstream primer in the step three is SEQNO.5, and the downstream primer is SEQNO.6.
Preferably, the saccharomyces cerevisiae inducible promoter in the step of performing is a galactose promoter GAL, a hexose transport vector or an alcohol dehydrogenase II.
Preferably, in the first step, a useless sequence can be introduced by overlap extension PCR as needed, so that the distance between two DSBs can be adjusted.
Preferably, the expression cassette of the I-SceI gene in the step comprises a galactose promoter, an I-SceI endonuclease and a KanMX/hygromycin/nourseothricin screening marker.
Specifically, the method comprises the following steps:
a method for constructing a double DSB model in yeast comprises the following steps:
disclosed is a plasmid which is constructed with an expression cassette for an I-SceI gene (GAL-I-SceI) under the control of a galactose promoter (GAL) and which is supplemented with a necessary resistance selection gene (HYG, KanMX, etc.). I-SceI is a homing endonuclease encoded by an intron and comprises 235 amino acids. The endonuclease can specifically recognize a DNA sequence (TAGGGATAACAGGGGTAAT) containing 18 asymmetric nucleotides, and cuts the DNA at a site containing the base sequence to form DSB. There are many similar endonucleases, which can be flexibly selected according to the needs.
Secondly, the GAL-I-SceI gene sequence is amplified through PCR, and DNA sequences (TAGGGATAACAGGGGTAAT) recognized by I-SceI enzyme and homology arms required by genome integration are respectively added at the two ends of designed PCR upstream and downstream primers. The insertion positions of the two I-SceI enzyme recognition sequences were adjusted as necessary to generate double DSBs with different distances apart. For efficient GAL-I-SceI integration into the genome of the target organism at a specific site, the length of the homology arms should be greater than 80 bp.
Thirdly, integrating the GAL-I-SceI gene sequence obtained by PCR amplification to a saccharomyces cerevisiae genome by using a chemical conversion method or other conversion methods, obtaining a successfully integrated strain by using a screening marker gene, and designing a verification primer to verify whether the construction is successful.
And a yeast containing GAL or other promoter controlled to express I-SceI gene, wherein when the promoter is activated, the I-SceI endonuclease can be expressed in a large amount, and the I-SceI recognition sites placed at both ends in advance are recognized and cut, so that double DSB is generated.
More specifically, the preparation and detection are as follows:
a construction method of a yeast fixed-point double-DSB synchronous induction model comprises the following specific steps:
designed upstream and downstream primers F1 and F2, I-SceI recognition sites were added to both ends of the primers, and an expression cassette of the I-SceI gene controlled by a galactose promoter (GAL) and a KanMX selection gene (GAL-I-SceI-KanMX) were amplified from plasmid pGSKU using F1 and F2. The spacing distance between two DSBs designed by the primer is 3620, and a spacing sequence (a DNA fragment without genetic effect between genes) can be introduced by overlap extension PCR according to the experimental needs, so that the spacing distance between the two DSBs is adjusted. Sequence F1: TTTCAGAGGTCGCCTGACGCTAGGGATAACAGGGTAATATACGCAAACCGCCTCTCCC, F2 sequence: TCCCAATTTTTCAGTTGAAAATAGGGATAACAGGGTAATGTCACCAAAGAACCAAGGGG, respectively;
pGSKU is a publicly available plasmid, and the expression cassette of the I-SceI gene is a target fragment directly amplified from the pGSKU plasmid. pGSKU map joining: https:// www.addgene.org/72243/.
Fastpfu dna polymerase reaction system: ddH2O15.5. mu.l, 1. mu.l of the upstream primer, 1. mu.l of the downstream primer, 0.5. mu.l of FastpfufuDNA polymerase, 1. mu.l of dNTP (2.5mM), 10 XPfuffer 5. mu.l, 1. mu.l of DNA template.
Fastpfu dna polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
And designing second primers F2 and R2 to carry out second PCR amplification, so that the homology arms of the upstream primer and the downstream primer are 70-80 bp. F2: ATCAAATTCGATGACTGGAAATTTTTTGTTAATTTCAGAGGTCGCCTGACGC; r2: ATGAAAAGCCGGTTCCGGCGCTCTCACCTTTCCTTTTTCTCCCAATTTTTCAGTTGAAA. The insertion site designed by the primer is the position of chromosome III 91055-one 91143, and can be changed according to different experimental requirements.
ddH reaction System of Fastpfu DNA polymerase2O15.5. mu.l, upstream1. mu.l of primer, 1. mu.l of reverse primer, 0.5. mu.l of FastpfufuDNA polymerase, 1. mu.l of dNTP (2.5mM), 10 XPfuffer 5. mu.l of DNA template, 1. mu.l.
Fastpfu dna polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
Thirdly, integrating the GAL-I-SceI gene sequence obtained by PCR amplification to a saccharomyces cerevisiae genome by using a chemical conversion method or other conversion methods, obtaining a successfully integrated strain by using a screening marker gene, and designing a verification primer to verify whether the construction is successful. An upstream primer: CAAGTAATTGGTTGTTTGGC, respectively; a downstream primer: TAGCCAGTTTGTTGAAAGCTTGGT are provided. Wherein, the control group (W): a band cannot be amplified by using the verification primer; experimental groups (1-5): 3620bp fragments can be amplified by using the primers; the results are shown in FIG. 3;
and a yeast containing GAL or other promoter controlled to express I-SceI gene, wherein when the promoter is activated, the I-SceI endonuclease can be expressed in a large amount, and the I-SceI recognition sites placed at both ends in advance are recognized and cut, so that double DSB is generated.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Sequence listing
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Construction method of <120> yeast fixed-point double-DSB synchronous induction model
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Claims (7)
1. A construction method of a yeast fixed-point double-DSB synchronous induction model is characterized by comprising the following steps: the method comprises the following steps:
the method comprises the steps of designing an upstream primer and a downstream primer, adding I-SceI recognition sites at two ends of the primers, and amplifying an expression cassette of an I-SceI gene controlled by a saccharomyces cerevisiae inducible promoter and a KanMX screening gene GAL-I-SceI-KanMX from a plasmid pGSKU by using the upstream primer and the downstream primer;
designing a second section of primer for second PCR amplification, so that the homology arms of the upstream primer and the downstream primer are 70-80 bp;
thirdly, integrating the GAL-I-SceI gene sequence obtained by PCR amplification to a saccharomyces cerevisiae genome by using a conversion method, obtaining a successfully integrated strain by using a screening marker gene, and designing a verification primer to verify whether the construction is successful;
and a yeast containing GAL or other promoter controlled to express I-SceI gene, wherein when the promoter is activated, the I-SceI endonuclease can be expressed, and the I-SceI recognition sites placed at both ends in advance are recognized and cleaved, so that double DSB is generated.
2. The method for constructing the yeast site-specific double-DSB synchronous induction model according to claim 1, wherein the method comprises the following steps: the method comprises the steps that an upstream primer is SEQ NO.1, and a downstream primer is SEQ NO. 2;
DNA polymerase reaction System: ddH2O15.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, DNA polymerase 0.5. mu.l, dNTP 1. mu.l at 2.5mM, 10 XPfuuffer 5. mu.l, DNA template 1. mu.l;
DNA polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
3. The method for constructing the yeast site-specific double-DSB synchronous induction model according to claim 1, wherein the method comprises the following steps: in the step II, the upstream primer is SEQ NO.3, and the downstream primer is SEQ NO. 4;
DNA polymerase reaction System: ddH2O15.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, DNA polymerase 0.5. mu.l, dNTP 1. mu.l at 2.5mM, 10 XPfuuffer 5. mu.l, DNA template 1. mu.l;
DNA polymerase program settings: the pre-denaturation at 96 ℃ for 2min is carried out for 1 cycle, the denaturation at 94 ℃ for 30s, the annealing at 56 ℃ for 60s and the extension at 72 ℃ for 1500bp/min are carried out for 32 cycles, and the extension at 72 ℃ for 7min is carried out for 1 cycle.
4. The method for constructing the yeast site-specific double-DSB synchronous induction model according to claim 1, wherein the method comprises the following steps: the third step is that the upstream primer is SEQ NO.5, and the downstream primer is SEQ NO. 6.
5. The method for constructing the yeast site-specific double-DSB synchronous induction model according to claim 1, wherein the method comprises the following steps: the saccharomyces cerevisiae inducible promoter in the step is a galactose promoter GAL, a hexose transport vector or an alcohol dehydrogenase II.
6. The method for constructing the yeast site-specific double-DSB synchronous induction model according to claim 1, wherein the method comprises the following steps: the step includes introducing a section of useless sequence through overlap extension PCR according to needs, and therefore the distance between two DSBs is adjusted.
7. The method for constructing yeast site-specific double DSB synchronous induction model according to any one of claims 1 to 6, wherein: the expression cassette of the I-SceI gene in the step comprises a galactose promoter, an I-SceI endonuclease and a KanMX/hygromycin/nourseothricin screening marker.
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