CN111088251B - Gene expression cassette and application thereof in Cre-lox recombination efficiency detection - Google Patents

Gene expression cassette and application thereof in Cre-lox recombination efficiency detection Download PDF

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CN111088251B
CN111088251B CN201911369104.9A CN201911369104A CN111088251B CN 111088251 B CN111088251 B CN 111088251B CN 201911369104 A CN201911369104 A CN 201911369104A CN 111088251 B CN111088251 B CN 111088251B
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施金秀
罗燕
王娟
叶知晟
蓝田
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Yunzhou Biotechnology Guangzhou Co ltd
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Abstract

The invention relates to the field of molecular biology, in particular to a gene expression cassette and application thereof in Cre-lox recombination efficiency detection. The gene expression cassette comprises a first promoter, a first lox site, a reporter element, a second lox site, a target gene and a third terminator which are connected in sequence; wherein the first lox site and the second lox site are co-directional; the reporter element comprises a second promoter, a second terminator and a reporter gene in sequence, and the second promoter, the second terminator and the reporter gene are reversely inserted into the gene expression cassette in situ; the reporter element further comprises a first terminator, the first terminator being located between the second promoter and the second terminator or between the second terminator and the reporter gene.

Description

Gene expression cassette and application thereof in Cre-lox recombination efficiency detection
Technical Field
The invention relates to the field of molecular biology, in particular to a gene expression cassette and application thereof in Cre-lox recombination efficiency detection.
Background
Cre-lox technology is a site-specific recombination technology discovered from the P1 bacteriophage in the 80's of the 20 th century. The technology can realize deletion, inversion, insertion, translocation and the like of a target fragment through the interaction of Cre recombinase and lox sites. The technology does not need any auxiliary factor, can act on DNA substrates with various structures, such as linear, circular or even supercoiled DNA and the like, is simple, rapid and efficient to operate, and is widely applied to researches such as gene knockout, insertion, turnover, translocation and the like of eukaryotes and prokaryotes. Currently, the hottest field of application of the Cre-lox system is gene targeting, which has proven to be the most useful tool for genetic manipulation of mammalian cells and mice, and the combination of this system and gene targeting provides a means for achieving conditional gene knockout or activation.
The Cre-lox system comprises two components: cre recombinase and lox recombination sites. The Cre recombinase is discovered from bacteriophage P1 in 1981, belongs to lambda int enzyme supergene family, has a coding region sequence with full length of 1029bp, codes 38kDa monomer protein consisting of 343 amino acids, and structurally consists of 4 subunits and two structural domains: the larger carboxy (C-terminal) domain and the smaller amino (N-terminal) domain specifically recognize and mediate recombination reactions between lox sites. The lox recombination sites are derived from the P1 phage, have a total length of 34bp, and consist of asymmetric 8bp spacers and inverted repeats flanked by 13 bp. Wherein, the two inverted repeat sequences are the specificity recognition site and the binding domain of Cre recombinase, and the middle 8bp spacing sequence determines the direction of lox site. The lox sites most commonly used are loxP (lox of X-over P1), and the Cre recombinase mediated recombination between two loxP sites is a dynamic reversible process, which can mediate deletion, inversion, insertion and ectopic of fragments according to the orientation and position of two loxP sites. One potential limitation of using loxP sites is that when there are more than two loxP sites, recombination between loxP sites cannot be controlled, intramolecular events occur more frequently than intermolecular events, and recombination reaction between any two loxP sites is likely to occur, which is very disadvantageous for some experiments requiring strict control of recombination reaction, and in order to overcome this defect, lox site mutants having various functions are generated by mutating loxP sites, and commonly used lox mutants are lox2272, lox71, lox66, etc., and systems such as FLEX and lox66/lox71, etc. are formed according to their different applications.
The Cre-loxP system can be used for transfecting target cells by Cre recombinase expression plasmids on the cellular level, excising resistance marker genes by identifying loxP sites, crossing a recombination heterozygote mouse and a Cre transgenic mouse on the individual level, and screening a progeny mouse to obtain a conditional knockout mouse with a deleted exogenous marker gene, so that the Cre-loxP system is the most commonly used Cre-loxP system. The LoxP-Stop-LoxP system utilizes a Cre-mediated conditional gene expression system, which contains 3 LSL elements of SV40 polyA sequences, and the conventional expression structure of the LSL elements is Promoter-LSL-GOI-polyA. In the absence of Cre recombinase, LSL is effective to terminate transcription of a downstream gene of interest, whereas in the presence of Cre LSL is excised to transcribe the downstream gene of interest. When the GOI is fluorescent protein, the recombination efficiency of the Cre-loxP system can be conveniently judged by detecting the fluorescence expression intensity. However, when the GOI is other non-luminescent functional protein, the recombination efficiency can only be detected by the protein expression level, which not only is the operation tedious, time consuming and cost increasing, but also the detection result has larger error compared with the actual situation, so that a better expression structure needs to be designed to solve the problem.
Disclosure of Invention
The Promoter-LSL-GOI-PolyA expression structure adopted by the traditional method has obvious defects: when the GOI is non-fluorescent protein, the recombination efficiency of Cre-lox cannot be visually observed and detected, and the conventional protein detection method is complicated and has relatively large error, so that the application range of the system is greatly limited. In order to solve the above problems, the present invention provides the following technical solutions:
the invention relates to a gene expression cassette, which comprises a first promoter, a first lox site, a reporter element, a second lox site, a target gene and a third terminator which are connected in sequence;
wherein the first lox site and the second lox site are co-directional;
the reporter element comprises a second promoter, a second terminator and a reporter gene in sequence, and the second promoter, the second terminator and the reporter gene are reversely inserted into the gene expression cassette in situ;
the reporter element further comprises a first terminator, the first terminator being located between the second promoter and the second terminator or between the second terminator and the reporter gene.
The gene expression cassette can intuitively obtain the recombination efficiency condition by detecting the fluorescence intensity of the cyclization product, particularly under the condition that the expression product of the target gene is fluorescent protein, the recombination efficiency of a Cre-lox system can be directly obtained by observing the condition of the report gene expression product in the excised ring without detecting the expression condition of the protein, and the gene expression cassette has the characteristics of simplicity, rapidness and high efficiency.
The invention also relates to a gene expression cassette into which a target gene is not inserted.
The invention also relates to a vector and a cell containing the expression cassette and application of the expression cassette in Cre-lox recombination efficiency detection.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the structure of a gene expression cassette provided in one embodiment of the present invention;
FIG. 2 shows the fluorescence expression at different time intervals after recombination by Cre-loxP system in an embodiment of the present invention; a1, A2 and A3 are all carriers which are singly transferred into 293T cells for 72 hours; b1, B2 and B3 are carriers and pRP [ Exp ] -EF1a > Cre cotransformed into 293T cells for 24 hours; c1, C2 and C3 are all vectors and pRP [ Exp ] -EF1a > Cre co-transferred into 293T cells for 48 hours; d1, D2 and D3 are vectors and pRP [ Exp ] -EF1a > Cre cotransformed into 293T cells for 264 hours;
pictures are all 100x, A1, B1, C1 and D1 are all white light channel imaging, and the exposure time is 10 ms; a2, B2, C2 and D2 are GFP channel images, and the exposure time is 200 ms; a3, B3, C3 and D3 are all RFP channel imaging, and the exposure time is 200 ms.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to a gene expression cassette, which comprises a first promoter, a first lox site, a reporter element, a second lox site, a target gene and a third terminator which are connected in sequence;
wherein the first lox site and the second lox site are co-directional;
the reporter element comprises a second promoter, a second terminator and a reporter gene in sequence, and the second promoter, the second terminator and the reporter gene are reversely inserted into the gene expression cassette in situ;
the reporter element further comprises a first terminator, the first terminator being located between the second promoter and the second terminator or between the second terminator and the reporter gene.
The reporter element can form a circularization element which is formed by sequentially connecting the second promoter, the reporter gene and the second terminator after the Cre enzyme cuts the first lox site and the second lox site, and the circularization element can express the reporter gene.
A34 bp special site sequence of Lox site is composed of 8bp asymmetric sequence (determining the direction of loxP sequence) and two 13bp symmetric sequences at both sides of the asymmetric sequence. "the first lox site and the second lox site are homodromous" means that the asymmetric region of 8bp is homodromous.
For convenience, in some embodiments, the following abbreviations are used in the present disclosure:
a first Promoter (Promoter1), a first lox site (L), a second lox site (L), a target Gene (GOI), and a third terminator (PolyA 2);
a second Promoter (Promoter2), a first terminator (S), a second terminator (PolyA1), a Reporter gene (Reporter).
In the present invention, anti { } indicates that the sequence is in the opposite direction to the transcription of the gene.
The invention discloses a new method for designing a carrier structure capable of detecting Cre-lox recombination efficiency by observing fluorescence intensity, and a specific embodiment is shown in figure 1: promoter1-L-anti { Promoter2} -S-anti { P olyA1} -anti { Reporter } -L-GOI-PolyA 2. To enhance the subsequent detection of Reporter, Pro moter2 can select a constitutive strong promoter.
When Cre recombinase does not exist in the system, the downstream GOI can not be expressed due to the termination effect of the first terminator (S); in the case of Cre expression, the sequence between the two lox sites is deleted and Promoter1 initiates expression of the GOI. At the same time, the sequence between the two loxs that were excised cyclized itself to Promoter2-Reporter-PolyA1, forming a plasmid-like loop structure that exists extrachromosomally. The structure is closed ring-shaped and has certain stability, so that the Reporter can have a certain amount of free expression in cells, the ring-shaped DNA structure free outside a chromosome is gradually reduced along with the division of the cells, the protein product of the Reporter is gradually reduced, and the fluorescence intensity is gradually weakened, so that the fluorescence expression cannot be observed.
It should be noted that fig. 1 is not a limitation of the present invention. Since the Promoter2-Reporter-PolyA1 is circular, the first terminator (S) can be inserted upstream or downstream of PolyA 1.
In some embodiments, the expression product of the gene of interest is any luminescent or non-luminescent functional protein.
In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., polyadenylation signals, poly-U sequences, etc.), are also included in the gene expression cassettes of the invention.
A "terminator" comprises a DNA sequence which is involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in some embodiments, a termination signal is contemplated that terminates the production of an RNA transcript. In vivo, a terminator may be necessary to obtain the desired level of information. In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow specific site cleavage of the new transcript to expose a polyadenylation site. It signals a specialized endogenous polymerase to add a stretch of about 200A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA appear to be more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, preferably, the terminator comprises a signal to cleave with RNA, more preferably, the terminator signal facilitates polyadenylation of the message. Terminator and/or polyadenylation site elements can be used to increase the level of information and minimize read-through from the cassette to other sequences.
Terminators contemplated for use in the present invention include any known transcription terminator described herein or known to those of skill in the art, including, but not limited to, for example, gene termination sequences. In some embodiments, the termination signal may be absent of a transcribable or translatable sequence, such as due to a sequence truncation. In some embodiments, the first terminator, the second terminator, and the third terminator are independently selected from the group consisting of an SV40 terminator, an hGH terminator, a BGH terminator, and an rbGlob terminator.
In some embodiments, the first terminator has a copy number ≧ 3, such as 4, 5, 6, 7, 8, or more. Preferably, the first terminator is the 3 × SV40 terminator.
A "promoter" is a DNA sequence that directs the binding of RNA polymerase and thereby initiates RNA synthesis. The promoters used in the present invention allow expression in a wide variety of cell and tissue types; or may be a cell-free specific promoter, or may be a "cell-specific", "cell type-specific", "cell lineage-specific" or "tissue-specific" promoter, which allows expression in a limited variety of cell and tissue types, respectively.
In particular embodiments, it may be desirable to use cell, cell type, cell lineage, or tissue specific expression control sequences to achieve cell type specific, cell lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a nucleic acid encoding a polypeptide only in a subpopulation of cell types, cell lineages, or tissues, or at a particular developmental stage).
Illustrative examples of tissue-specific promoters include, but are not limited to: b29 promoter (B cell expression), run transcription factor (CBFa2) promoter (stem cell specific expression), CD14 promoter (monocyte expression), CD43 promoter (leukocyte and platelet expression), CD45 promoter (hematopoietic cell expression), CD68 promoter (macrophage expression), CYP4503a4 or ALB promoter (hepatocyte expression), myodesmin promoter (muscle cell expression), elastase I promoter (pancreatic acinar cell expression), endoglin promoter (endothelial cell expression), fibroblast specific protein I promoter (FSPl) promoter (fibroblast expression), fibronectin promoter (fibroblast expression), fms-related tyrosine kinase I fltl promoter (endothelial cell expression), Glial Fibrillary Acidic Protein (GFAP) promoter (astrocyte expression), insulin promoter (pancreatic cell expression), Integrin-alpha-2 b (ITGA2B) promoter (megakaryocytes), intracellular adhesion molecule 2(ICAM-2) promoter (endothelial cells), interferon-beta (IFN-beta) promoter (hematopoietic cells), keratin 5 promoter (keratinocyte expression), Myoglobin (MB) promoter (muscle cell expression), myogenic differentiation I (MYOD1) promoter (muscle cell expression), nephroprotein promoter (podocyte expression), bone gamma-carboxyglutamic acid protein 2(OG-2) promoter (osteoblast expression), 3-keto acid CoA transferase 2B (Oxct2B) promoter (haploid sperm cell expression), surface-activated protein B (SP-B) promoter (lung cell expression), synaptoprotein promoter (nerve cell expression), Wiskott-Aldrich syndrome protein (WASP) promoter (hematopoietic cell expression).
In some embodiments, the first promoter and the second promoter are both cell-free specific promoters. Exemplary cell-free specific promoters include, but are not limited to, Cytomegalovirus (CMV) very early promoter, viral simian virus 40(SV40) (e.g., early or late), moloney murine leukemia virus (MoMLV) LTR promoter, Rous Sarcoma Virus (RSV) LTR, Herpes Simplex Virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pll promoter of vaccinia virus, elongation factor l- α (EFla) promoter, early growth response i (egrl), ferritin H (ferh), ferritin l (ferl), 3_ glyceraldehyde phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5(HSPA5), heat shock protein 90- β member 1(HSP90B1), heat shock protein 70kDa (HSP70), β -kinesin (β -KIN), human R0SA26 locus (irs et al, ion et al, seq. (2007) Nature Biotechnology25,1477-1482), the ubiquitin C promoter (UBC), the phosphoglycerate kinase-1 (PGK) promoter, the cytomegalovirus enhancer/chicken β -actin (CAG) promoter, and the β -actin promoter. In some embodiments, the cell-free specific promoter is preferably selected from the group consisting of CMV promoter, EFS promoter, miniCMV promoter, EFl-a promoter, and PGK promoter. The promoter without cell specificity can ensure that the gene expression cassette has better universality and expression efficiency. In some embodiments, the promoter (preferably the second promoter) is a constitutively strong promoter.
In some embodiments, the first lox site and the second lox site are independently selected from the group consisting of a wild-type loxP site and a mutant lox site.
In some embodiments, the lox site of the mutation is selected from the group consisting of lox71, lox75, lox44, lox66, lox76, lox43, lox72, lox78, lox65, lox511, lox512, lox514, lox5171, lox2272, loxM2, loxM3, loxM7, and loxM 11.
The reporter gene can be selected from metabolic markers, catalytic reporter genes, antibiotic markers, antibiotic resistance genes, herbicide resistance genes, auxotrophic reporter genes, compound detoxification enzyme genes, and carbohydrate metabolism enzyme selection marker genes that are well known to those skilled in the art;
in some preferred embodiments, for ease of observation and detection, the expression product of the reporter gene is a substance that can self-emit light or produce a color change by catalyzing a substrate reaction, or can cause a substrate to emit light or produce a color change by catalyzing a substrate reaction, or produce emitted light or produce a color change upon irradiation with excitation light. Such substances typically include fluorescent protein, luciferase and LacZ. Both the fluorescent protein and the luciferase are luminescent proteins, and the expression of fluorescence can be detected by a camera or the like. Fluorescent proteins work by absorbing light of one color (excitation) and then emitting a different color (emission) of lower energy light. In contrast, luciferase (and other bioluminescent enzymes) emit light by catalyzing a chemical reaction of a substrate (i.e., luciferin). Unlike the two labels above, LacZ does not emit light. The product of the LacZ gene, beta-galactosidase, catalyzes the conversion of X-gal to an opaque blue compound similar to indigo.
Further, the fluorescent protein may be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein. The green fluorescent protein can adopt common GFP, and can also adopt modified GFP genes, such as enhanced GFP gene EGFP and the like; the blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP and the like; the yellow fluorescent protein can be selected from EYFP, Ypct, PhiYFP and the like; the orange fluorescent protein can be selected from mKO, mOrange, mBanana and the like; the red fluorescent protein can be selected from TagRFP, mRuby, mCherry, mKate and the like.
According to still another aspect of the present invention, the present invention also relates to a gene expression cassette obtained by replacing a gene of interest in the gene expression cassette as described above with a gene of interest insertion site.
The target gene insertion site can be selected from enzyme cutting sites common in the field, so that the target gene can be conveniently inserted.
The present invention also provides a vector comprising a gene expression cassette as described above.
The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. The vector is preferably derived from a virus. The vector preferably contains the essential sequences required for packaging and delivery to the cell. It is desirable that the vector be composed in part of enveloped or non-enveloped viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40).
The invention also provides a cell, the genome of which contains the gene expression cassette.
The cell refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. The host cell is preferably a eukaryotic cell, more preferably a mammalian cell.
In some embodiments, the cell further has a recombinase Cre and/or a recombinase Cre expression system.
The invention also claims transgenic organisms, all or part of the cells (e.g. cells of a specific organ or a specific tissue) of which contain in their genome the gene expression cassettes as described above.
In some embodiments, the transgenic organism is a fertilized egg or embryo that has not developed into an individual organism.
In some embodiments, the transgenic organism is a plant, such as arabidopsis thaliana or tobacco.
In some embodiments, the transgenic organism is an animal.
In some embodiments, the transgenic organism is an insect (e.g., caenorhabditis elegans), a fish (e.g., zebrafish), or a mammal (e.g., a human, rat, or mouse).
According to another aspect of the present invention, the present invention also relates to a Cre-lox recombination efficiency detection method, comprising:
a) providing a cell as described above and contacting the genome of said cell with recombinase Cre;
b) detecting the expression condition of the reporter gene in the cell, wherein the expression intensity of the reporter gene is an indication of high Cre-lox recombination efficiency.
Embodiments of the present invention will be described in detail with reference to examples.
Examples
As shown in FIG. 1, the embodiment overcomes the defects of the conventional Promoter-LSL-GOI-PolyA expression structure by designing a novel Promoter1-L-anti { Promoter2} -S-anti { PolyA1} -anti { Reporter } -L-GOI-PolyA2 structure, obtains a simpler, faster, efficient and wider-application-range method for detecting Cre-lox recombination efficiency, and verifies the method at the cell level.
1. Vector construction:
1.1 an LR reaction is carried out on an intermediate vector pDOwn-LoxP-anti { EFS } -3xSV40-anti { BGH pA } -anti { EGFP } -LoxP, pUP-CMV and pTail-mCherry and a Destination vector pUC19(mini), Des3d.null (G to A) to construct a vector: pRP [ Exp ] -CMV > Loxp-anti { EFS } -3XS 40pA-anti { BGH pA } -anti { EGFP } -Loxp > mCherry, namely Promoter1 is a CMV Promoter, L is LoxP, Promoter2 is an EFS Promoter, PolyA1 is BGH pA, Reporter is an EGFP green fluorescent Reporter, and GOI is an mCherry red fluorescent Reporter.
2. Cell level verification of the recombination efficiency of Cre-loxP system:
2.1 taking 1ug of the vector to transiently transfect 293T cells, and observing whether the fluorescence of the cells has leakage expression;
2.2 1ug of the above vector was co-transfected with 1ug of pRP [ Exp ] -EF1a > Cre vector into 293T cells.
2.3 observe and record the fluorescence expression in different time periods, and the result is shown in FIG. 2.
2.4 the experimental results show that: (A) after pRP [ Exp ] -CMV > Loxp (r) -anti { EFS } -3XSV40 pA-anti { BGH pA } -anti { EGFP } -Loxp (r) > mCherry singly transferred 293 cells for 72h, green and red fluorescence expression can not be observed, which indicates that no cyclization can be carried out between loxP sites and EGFP can not be expressed under the condition of no Cre, so green fluorescence can not be observed; the presence of 3 × SV40pA also terminated transcription of mCherry by CMV, so no expression of red fluorescence was observed. (B) The vector and Cre enzyme expression vector pRP [ Exp ] -EF1a > Cre co-transfer and obvious red and green fluorescence expression begins to appear 24 hours later. (C) The red and green fluorescence reached the brightest after 48 hours of co-rotation. (D) After 264 hours of co-rotation, no red and green fluorescence could be observed, indicating that the vector was gradually lost in 293T cells with cell division and was completely lost after 264 hours.
The above examples illustrate that the gene expression cassette provided by the present invention can simply and rapidly detect the recombination efficiency of Cre-loxP.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (13)

1. A gene expression cassette comprising a first promoter, a first lox site, a reporter element, a second lox site, a gene of interest, and a third terminator, connected in sequence;
wherein the first lox site and the second lox site are co-directional;
the reporter element comprises a second promoter, a second terminator and a reporter gene in sequence, and the second promoter, the second terminator and the reporter gene are reversely inserted into the gene expression cassette in situ;
the reporter element further comprises a first terminator, the first terminator being located between the second promoter and the second terminator or between the second terminator and the reporter gene.
2. The gene expression cassette of claim 1, wherein the first, second and third terminators are independently selected from the group consisting of SV40 terminator, hGH terminator, BGH terminator and rbGlob terminator.
3. The gene expression cassette of claim 2, the first terminator having a copy number of 3 or 4.
4. The gene expression cassette of claim 1, wherein the first and second promoters are both cell-free specific promoters.
5. The gene expression cassette of claim 4, wherein the cell-free specific promoter is selected from the group consisting of CMV promoter, EFS promoter, miniCMV promoter, EFl-a promoter and PGK promoter.
6. The gene expression cassette of claim 1, wherein the first lox site and the second lox site are independently selected from the group consisting of a wild-type loxP site and a mutant lox site.
7. The gene expression cassette according to claim 6, wherein the lox site of the mutation is selected from the group consisting of lox71, lox75, lox44, lox66, lox76, lox43, lox72, lox78, lox65, lox511, lox512, lox514, lox5171, lox2272, loxM2, loxM3, loxM7 and loxM 11.
8. The gene expression cassette according to claim 1, wherein the expression product of the reporter gene is a substance that emits light or changes color by itself or by the reaction of a catalytic substrate or by the irradiation of excitation light.
9. A gene expression cassette, wherein a target gene in the gene expression cassette according to any one of claims 1 to 8 is replaced with a target gene insertion site.
10. A vector comprising the gene expression cassette of any one of claims 1 to 9.
11. A cell comprising the gene expression cassette of any one of claims 1 to 8 in its genome.
12. The cell of claim 11, further comprising a recombinase Cre and/or a recombinase Cre expression system.
A Cre-lox recombination efficiency detection method, comprising:
a) providing the cell of claim 11 and contacting the genome of the cell with recombinase Cre;
b) detecting the expression condition of the reporter gene in the cell, wherein the expression intensity of the reporter gene is an indication of high Cre-lox recombination efficiency.
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