CN112921052B - In vivo cell proliferation marker and tracer system and application thereof - Google Patents

In vivo cell proliferation marker and tracer system and application thereof Download PDF

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CN112921052B
CN112921052B CN201911242892.5A CN201911242892A CN112921052B CN 112921052 B CN112921052 B CN 112921052B CN 201911242892 A CN201911242892 A CN 201911242892A CN 112921052 B CN112921052 B CN 112921052B
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周斌
刘秀秀
何灵娟
蒲文娟
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Center for Excellence in Molecular Cell Science of CAS
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Abstract

The present invention relates to polynucleotide sequences comprising a coding sequence for a chimeric recombinase, a fragment comprising a cell proliferation factor gene, a coding sequence for a first recombinase, a coding sequence for an estrogen receptor ER ligand binding domain, and a recognition site for a second recombinase. The present invention also provides the polynucleotides product, nucleic acid construct, host cell, etc. containing the polynucleotides sequence. The invention also provides in vivo proliferative cell markers or tracers methods using the polynucleotide products, nucleic acid constructs and host cells of the invention. The invention provides a proliferation cell marking system through the artificial modified chimeric recombinase, which realizes continuous long-time cell marking and tracing. The invention tracks the proliferation of various cells in the body for the first time, and has important significance for knowing the dynamic change of various cell groups in the body.

Description

In vivo cell proliferation marker and tracer system and application thereof
Technical Field
The present findings relate to cell proliferation markers and tracer systems and uses thereof.
Background
Proliferation of cells in vivo is a dynamic, unstable process, proliferation of cells of the same type within the same time is different, capturing the state of proliferation of cells in vivo is critical to understanding both biophysical and pathological processes, and thus capturing proliferation of cells in vivo has been a hotspot of research in the field of biology. Traditional methods for capturing cell proliferation mainly include proliferation molecular marker staining and incorporation of DNA analogs and isotopes. However, all three have the defects that the proliferation molecular markers are used for dyeing to capture the proliferation of cells in a short time; secondly, the cell proliferation can only be studied within a certain period of time by using the DNA analogue incorporation, because the long-term incorporation of the DNA analogue is very likely to influence the normal proliferation of cells; however, the isotope incorporation method reduces the influence on normal proliferation of cells, but introduces a problem of difficulty in detection.
There have been studies in the art of in vivo proliferation cell tracing using genetic lineage tracing techniques. For example, researchers have used Ki67 cell proliferation markers as promoters to initiate inducible homologous recombinases to homologous recombine corresponding reporter genes and label cells, i.e., mice obtained by hybridization of Ki67-CreER tool mice with corresponding sustained open reporter genes. When the cell expresses Ki67 and proliferates, the activation of the Ki67 promoter starts the subsequent expression of CreER, and simultaneously, the cell marks the corresponding fluorescence of the reporter gene by homologous recombination of LoxP locus of the corresponding reporter gene under the induction of tamoxifen (tamoxifen). Although this technique has a certain role in tracking proliferating cells in vivo, it has a significant drawback in that the system for in vivo tracking of proliferating cells using Ki67-CreER relies on two conditions that exist simultaneously, one is that the activity of Ki67 initiates the expression of CreER and the other is that tamoxifen (tamoxifen) which lets CreER enter the nucleus exists simultaneously in vivo. If a faster proliferating cell is tracked, then it is essentially captured during the time that tamoxifen acts on the body, but if a cell is actively driven by Ki67 during a subsequent time, but there is no tamoxifen available in the body to allow Cre to enter the nucleus, then it is not labeled with fluorescence and this signal will be missed when a slower proliferating cell is tracked because of its shorter time of action in the body. It is not practical to perform treatment of tamoxifen all the time during long-time tracking, on the one hand, the physiological state of mice is affected, and on the other hand, the efficiency problem of tamoxifen in inducing Cre into the nucleus is also certain. The study aims at establishing a set of genetic lineage tracing technology for tracing in vivo proliferation cells, capturing signals which are easy to be missed due to the transience of proliferation gene expression, and continuously reducing in vivo cell proliferation conditions for a long time.
Disclosure of Invention
In a first aspect, the invention provides a nucleic acid molecule selected from the group consisting of
(1) Nucleic acid molecule comprising a 5 'homology arm and a 3' homology arm, and a first recombinase coding sequence, an estrogen receptor ER coding sequence, and a recognition site for a second recombinase located between the 5 'homology arm and the 3' homology arm, said 5 'homology arm and 3' homology arm being capable of recombining sequences therebetween into a genome such that the sequences therebetween are co-expressed with a cell proliferation factor gene in the genome, and
(2) (1) the complement of the nucleic acid molecule.
In one or more embodiments, the polynucleotide sequence of the nucleic acid molecule is, in order from the 5 'end to the 3' end, a 5 'homology arm, a first recombinase coding sequence, a recognition site for a second recombinase, an estrogen receptor ER coding sequence, a recognition site for a second recombinase, and a 3' homology arm, the 5 'homology arm and the 3' homology arm being capable of recombining sequences therebetween to the 5 'or 3' end of the cell proliferation factor gene.
In one or more embodiments, the cell proliferation factor is Ki67 or PCNA.
In one or more embodiments, the nucleic acid sequence of the 5' homology arm is as shown in nucleotides 1-3000 of SEQ ID NO. 1.
In one or more embodiments, the nucleic acid sequence of the 3' homology arm is as shown in nucleotides 5128-8127 of SEQ ID NO. 1.
In one or more embodiments, the first and second recombinases are Cre and Dre or Dre and Cre, respectively, wherein the recognition site of Cre is LoxP and the recognition site of Dre is rox.
In one or more embodiments, the amino acid sequence of Dre is as shown in amino acids 1-356 of SEQ ID NO. 2.
In one or more embodiments, the amino acid sequence of Cre is shown in SEQ ID NO. 3. In one or more embodiments, the nucleic acid sequence of Cre is shown in SEQ ID NO. 1 at 3067-4099.
In one or more embodiments, the nucleic acid sequence of LoxP is shown in SEQ ID NO. 4.
In one or more embodiments, the nucleic acid sequence of rox is set forth in SEQ ID NO. 5.
In one or more embodiments, the amino acid sequence of the estrogen receptor ER is shown as amino acids 357-666 of SEQ ID NO. 2. In one or more embodiments, the nucleic acid sequence of the estrogen receptor ER is shown as nucleotides 4153-5085 of SEQ ID NO. 1.
The invention also provides a nucleic acid construct comprising a nucleic acid molecule as described herein.
The present invention also provides a recombinase system comprising (1) a nucleic acid molecule as described herein, and (2) optionally, a nucleic acid molecule encoding a fusion protein of a second recombinase and an estrogen receptor ER, (3) optionally, a nucleic acid molecule comprising the structure: from the 5 'end to the 3' end are, in order, a recognition site for the first recombinase, a termination sequence, a first recombination site and a marker coding sequence.
The invention also provides a recombinase system comprising (1) a nucleic acid construct as described herein, and (2) optionally a second nucleic acid construct having a polynucleotide sequence encoding a fusion protein of a second recombinase and an estrogen receptor ER, (3) optionally a third nucleic acid construct having a polynucleotide sequence comprising the structure: from the 5 'end to the 3' end are, in order, a recognition site for the first recombinase, a termination sequence, a recognition site for the first recombinase and a marker coding sequence.
The invention also provides a host cell comprising one or more selected from the group consisting of: (1) a polynucleotide sequence as described herein; (2) a nucleic acid construct as described herein; (3) a system as described herein.
The invention also provides a method of constructing a transgenic animal comprising:
will F 0 Generation of first animal and F 0 Generation of second animals or F 0 Mating with a third animal, homologous recombination in a offspring animal to obtain a transgenic animal comprising the first and second polynucleotide sequences or a transgenic animal comprising the first and third polynucleotide sequences, or
The three F are combined 0 Mating any two of the animals of the generation and then carrying out homologous recombination F 1 Substituted animals and third F 0 Mating of animals, at F 2 Homologous recombination occurs in the generation animal to obtain a transgenic animal comprising the first, second and third polynucleotide sequences.
In one or more embodiments, F 0 The genome of the first animal comprises a first polynucleotide sequence, which is the polynucleotide sequence of the nucleic acid molecule described herein, or which is the first recombinase coding sequence, the recognition site for the second recombinase, the estrogen receptor ER coding sequence, the recognition site for the second recombinase in this order from the 5 'end to the 3' end, and which is co-expressed with the cell proliferation factor gene in the genome.
In one or more embodiments, F 0 The genome of the second animal comprises a second polynucleotide sequence encoding a fusion protein of a second recombinant enzyme and an estrogen receptor ER.
In one or more embodiments, F 0 The genome of the third animal contains a third polynucleotide sequence which is a recognition site of the first recombinase, a termination sequence, a recognition site of the first recombinase and a marker coding sequence in sequence from the 5 'end to the 3' end.
In one or more embodiments, the animal is a mouse.
In one or more embodiments, the first and second recombinant enzymes, estrogen receptor ER, and cell proliferation factor are as described herein.
The invention also provides a method of constructing a transgenic animal comprising introducing and culturing any one, two or three of the first, second, third polynucleotide sequences into an animal cell, and selecting a transgenic animal comprising any one, two or three of the first, second, third polynucleotide sequences in its genome, wherein
The first polynucleotide sequence is a first recombinant enzyme coding sequence, a recognition site of a second recombinant enzyme, an estrogen receptor ER coding sequence and a recognition site of a second recombinant enzyme in sequence from a 5 'end to a 3' end, and the first polynucleotide sequence is co-expressed with a cell proliferation factor gene in a genome in the transgenic animal,
The second polynucleotide sequence encoding a fusion protein of a second recombinant enzyme and an estrogen receptor ER,
the third polynucleotide sequence is a recognition site of the first recombinase, a termination sequence, a recognition site of the first recombinase and a marker coding sequence in sequence from the 5 'end to the 3' end.
In one or more embodiments, the cell is an animal ES cell.
In one or more embodiments, the animal is a mouse.
In one or more embodiments, the first and second recombinant enzymes, estrogen receptor ER, and cell proliferation factor are as described herein.
The invention also provides a method of long-term cell labelling in vivo comprising labelling cells expressing a cell proliferation factor gene in an animal comprising first, second and third polynucleotide sequences in the presence of an inducer of interaction with an estrogen receptor ER, wherein,
the first polynucleotide sequence is a first recombinant enzyme coding sequence, a recognition site of a second recombinant enzyme, an estrogen receptor ER coding sequence and a recognition site of a second recombinant enzyme in sequence from a 5 'end to a 3' end, and the first polynucleotide sequence is co-expressed with a cell proliferation factor gene in a genome in the transgenic animal,
The second polynucleotide sequence encodes a fusion protein of a second recombinant enzyme and an estrogen receptor ER, and
the third polynucleotide sequence is a recognition site, a termination sequence, a first recombination site and a marker coding sequence of the first recombinase in sequence from the 5 'end to the 3' end.
In one or more embodiments, the animal is a mouse.
In one or more embodiments, the first and second recombinant enzymes, estrogen receptor ER, and cell proliferation factor are as described herein.
The invention also provides the use of a nucleic acid molecule, nucleic acid construct, system or host cell as described herein for long-term cell labeling or cell tracing.
The invention also provides a kit comprising a nucleic acid molecule, nucleic acid construct, system or host cell as described herein, and reagents required for knocking the nucleic acid molecule into the genome of the cell.
Drawings
FIG. 1 shows a conventional cell proliferation tracer technique and one embodiment of the invention. (a) Cartoon representation of Ki67 expression profile versus fate profile, ki67 is dynamically expressed at different time points from T1 to Tn, the fate profile of Ki67 may refer to capturing all expression of Ki67 from T1 to Tn. (b) DreeR is induced by tamoxifen to nuclear-cleave ER behind Cre, so that Ki67 can enter cells undergoing homologous recombination marker proliferation of a reporter gene once Cre is expressed, and cell proliferation can be tracked seamlessly over a long period of time. (c) The proliferation cells are tracked by using the traditional genetic lineage tracing technology, and due to the short action time window of tamoxifen, only cell proliferation can be detected in a short time.
FIG. 2 construction and validation of an exemplary chimeric recombinase in one embodiment of the invention. (a) Construction strategy of Ki67-CrexER knock-in mice is schematically shown. (b) Sectional immunofluorescent staining results of Ki67-CrexER gene knocked into the adult small intestine of mice. The results of staining for ESR and Ki67 and EdU, as well as statistics, showed that the expression profile of ESR was substantially consistent with the markers of proliferation. (c) Ki67-CrexER was mated with R26-GFP and tamoxifen (Tam) induction was performed in the adult, GFP and VE-Cad immunofluorescence stained two-photon confocal images of aortic sections obtained after 5 days. The statistical results show that the Ki67-CrexER marks endothelial cells present in pairs, indicating that the Ki67-CrexER knock-in mice capture proliferation activity of cells in vivo. The scale bar represents 100 μm in b and 500 μm in c. Each picture represents at least three biological replicates.
FIG. 3 shows a proliferative cell labeling and tracing (Protracker) method according to one embodiment of the invention. (a) The Ki67-CrexER is converted into a cartoon representation of Ki67-Cre under the induction of Dreer and tamoxifen. (b) Three mouse strains required for proliferation of cells were tracked using the methods described herein. (c) Tamoxifen induction and time profile of sample collection of the traditional methods and Protracker labelled proliferating cells. (d) The signals of proliferation of each tissue organ of adult mice in two days and four weeks of the traditional method and Protracker tracing are shown in the figure, the full tissue fluorescence and immunofluorescence result graphs after each tissue slice are shown, and the embedded small graph represents the bright field full tissue picture of the same tissue or organ. The upper graph in each graph is the result of the Ki67-Creer of the conventional method and the lower graph is the result of the Protracker system. The scale bar represents 1mm in the whole tissue picture and 100 μm in the slice fluorescence picture. Each picture represents five independent biological replicates.
FIG. 4 shows that Dreer and R26-GFP do not undergo mixed homologous recombination. The mice obtained by mating R26-DreeR with R26-GFP are induced by tamoxifen, and after four weeks, fluorescence of the whole tissue obtained by each tissue or organ is collected or GFP immunofluorescence is photographed after slicing. The embedded panels represent full tissue bright field photographs of the same tissue organ. The scale bar of the full tissue fluorescence plot of GFP represents 1mm, the other scale bars represent 100 μm, and each picture represents 5 biological replicates.
FIG. 5, ki67-CrexER; R26-GFP and Ki67-CrexER; R26-Dreer; R26-GFP showed substantially no signal leakage. (a, b) Ki67-CrexER 12 weeks old; R26-GFP and Ki67-CrexER; R26-Dreer; R26-GFP mice were stained for full tissue fluorescence and immunofluorescence of each tissue section. The scale bar was 1mm in the whole tissue and 100 μm in each tissue section. Each picture represents 5 individual biological replicates.
FIG. 6 proliferation of hepatocytes in adult liver. (a) Experimental strategy cartoon schematic diagram for tracing liver cell proliferation by using the method of the invention. (b) The liver is divided from the portal vein region to the central vein region into three cartoon representations of different metabolic regions, with arrows indicating the direction of blood flow. (c) Immunofluorescent staining results of GFP, GS and E-CAD from tamoxifen-induced Protracker mice on day 0, day 2 liver tissue sections. (d) Immunofluorescent staining results of GFP, GS and E-CAD in liver tissue sections of mice at weeks 2, 4, 6, 8, 10, 12 after induction with tamoxifen in protacker mice. (e) GFP per mm2 in different regions of liver lobule at different sampling time points + Is a cell count statistic of (a). 1. 2, 3 respectively represent Zone1 (E-CAD + )、Zone2(E-CAD - GS - )、Zone3(GS + ). Scale = 100 μm, each picture representing 5 individual biological replicates.
FIG. 7 proliferation of cardiomyocytes in adult hearts. (a) Cartoon schematic of lineage tracing of Ki67 expressing cells from day 1 to day n, green representing Ki67 expressed proliferation signal. (b) a cartoon representation of the proliferating cells being tracked seamlessly. (c) GFP, TNNI3 immunofluorescence staining results for heart sections of three month pedigree-traced ProTracker mice, with 1, 2 being left panels and partial area enlargement. (d) Fluorescent staining results for GFP, WGA of heart tissue sections of three month pedigree-traced protacker mice are shown with the left outlined region on the right side and GFP, TNNI3 immunofluorescent staining results for the same enlarged region on the right side.
FIG. 8, capture of nuclear division and cell division of cardiomyocytes in adult hearts. (a) Lineage tracing cartoon schematic showing Ki67 cell expression from day 1 to day n, green for proliferation signal of Ki67 expression and subsequent nuclear numbers can be performedAnd (5) analyzing. (b) Hearts of three month pedigree-spiked Protracker mice were isolated and Hoechst stained and counted for nuclei for GFP+ and GFP-cardiomyocytes. (c) 3D representation of GFP+ cardiomyocyte multilateral scan (xyz: 500X100 μm). (d) The enlarged multi-slice scan shows a GFP + The yellow arrow indicates the nucleus of the cardiomyocyte and the inset panel is a cartoon representation of the double-core myocyte. (e) The xy and yz magnified 3D plot of the partial region of c shows two immediately adjacent cardiomyocytes. (f) The enlarged multilateral scan shows that both immediately adjacent gfp+ cardiomyocytes are single core myocytes. Yellow arrows indicate cardiomyocyte nuclei, and hollow white arrows indicate non-cardiomyocyte nuclei of cardiomyocytes next to gfp+. The embedded panels are cartoon schematic of two adjacent single core myocytes. (g) GFP (Green fluorescent protein) + Is a count of the number of adjacent cardiomyocytes in the cardiomyocytes. The scale bar indicates 100. Mu.m. Each picture represents five independent biological replicates.
Detailed Description
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.
The invention aims to provide a genetic lineage tracing technology for tracing proliferation cells in vivo, which captures signals which are easily missed due to the transience of proliferation gene expression and reduces the proliferation of cells in vivo for a long time.
The mouse Ki67-CreER established using conventional genetic lineage tracing techniques was unable to track cell proliferation slowly over long periods of time due to the short window of in vivo action of tamoxifen, and was able to track cell proliferation only during the time period of tamoxifen action (fig. 1, c). Cell proliferation factor genes such as Ki67 are dynamically expressed in vivo, single proliferation marker staining and traditional lineage tracing methods can only capture proliferation expression profiles at a single time point, and only the fate map (rate map) of the cell proliferation factor gene can truly reflect the proliferation status of cells in vivo for a certain period of time (fig. 1, a). However, direct lineage tracing using Ki67-Cre is not feasible because all daughter cells are derived from the same fertilized egg. In order to be able to track cells that proliferate slowly over a long period of time in vivo, it is necessary to convert the inducible CreER under certain conditions into Cre that is expressed and then enters the nucleus.
To solve this problem, the present invention introduces a second recombination system Dre-rox independent of Cre-LoxP to engineer creers in the prior art pedigree tracing technique, adding a recognition site rox for Dre homologous recombinase at each end of their ER DNA sequence, and the engineered CreERT2 is transformed into Cre-rox-ERT2-rox, herein referred to as CrexER. When DreeR and Tamoxifen (Tamoxifen) exist in vivo simultaneously, tamoxifen induces Dre to enter the nucleus to generate homologous recombination of rox at two ends of ER DNA, crexER is changed from an inducible homologous recombinase into a homologous recombinase Cre which can enter the nucleus directly, so that proliferation specific genes in subsequent cells can directly identify reporter genes to mark the cells once the expression of Cre is started, as shown in (figure 1, b). The invention utilizes a cell proliferation tracking system (Cell proliferation tracker, protracker) to continuously track the expression of proliferation specific genes in cells for a long time.
The engineered tracer system of the invention comprises a nucleic acid molecule encoding a chimeric recombinase, the polynucleotide sequence of which is selected from the group consisting of: a polynucleotide sequence comprising a fragment of a cell proliferation factor gene, a coding sequence for a first recombinase, a coding sequence for an estrogen receptor ER, a recognition site for a second recombinase, and/or a complement of said polynucleotide sequence. In one embodiment, the polynucleotide sequence of the nucleic acid molecule is, in order from the 5 'end to the 3' end, a first fragment of the cell proliferation factor gene, a coding sequence for a first recombinase, a recognition site for a second recombinase, a coding sequence for an estrogen receptor ER, a recognition site for a second recombinase, and a second fragment of the cell proliferation factor gene. Preferably, the nucleic acid molecule of the invention is selected from (1) a nucleic acid molecule comprising a 5 'homology arm and a 3' homology arm, which are capable of recombining the sequences between them into the genome such that the sequences between them are co-expressed with a cell proliferation factor gene in the genome, and (2) a complementary sequence of the nucleic acid molecule of (1), and a first recombinase coding sequence, an estrogen receptor ER coding sequence, and a recognition site for a second recombinase located between the 5 'homology arm and the 3' homology arm.
The polynucleotides herein may be in DNA form or in RNA form. DNA forms include cDNA or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. In certain embodiments, the polynucleotide sequence is set forth in SEQ ID NO. 1.
"cell proliferation factor gene" or "cell proliferation specific gene" as used herein refers to a gene that is expressed only during cell proliferation. Cell proliferation as described herein includes, but is not limited to, mitosis, amitoses, meiosis, bidivision, and the like. In one embodiment, the cell proliferation factor is Ki67 and/or PCNA. Ki-67 is a proliferation cytokine which is currently used in a large amount, and Ki-67 is expressed at each stage of cell proliferation except for G0 phase. PCNA is an abbreviation for proliferating cell nuclear antigen (Proliferating Cell Nuclear Antigen) and is found only in normal proliferating cells and tumor cells.
Herein, the 5 'homology arm and the 3' homology arm may be fragments of the cell proliferation factor gene, as long as the fragments can be used for homology arms of homologous recombination. The fragment may comprise the promoter of the cell proliferation factor gene or be located after the promoter, or the fragment may be located before or in the 3'utr of the gene or comprise the 3' utr. Whereby expression of a sequence of interest (e.g., a chimeric recombinase described herein) is placed under the control of a specific promoter to cause expression in a specific tissue or organ or at a specific developmental stage. In one embodiment, the fragment is selected such that the cell proliferation factor gene is expressed or not after homologous recombination. In one embodiment, the fragment is selected such that expression of the cell proliferation factor gene is unaffected following homologous recombination. Fragments of the cytokine gene include first and second fragments, respectively, that serve as the 5 'homology arm and 3' homology arm required for the group of homologous recombination. In one embodiment, the first and second fragments are selected such that the sequence of interest is inserted downstream of the promoter of the cytokine gene. In one embodiment, the first and second fragments are selected such that the sequence of interest is inserted between the last exon and the 3' utr of the cytokine gene. In one or more embodiments, fragments of the cytokine gene are as set forth in SEQ ID NO. 1, 1-3000 and 5128-8127.
Suitable recombinases for use herein can be any recombinase, including a first recombinase and a second recombinase. The first and second recombinant enzymes herein may be different. In one embodiment, the first and second recombinant enzymes are Cre and Dre or Dre and Cre, respectively. The Cre recombinase herein may be a Cre recombinase known in the art, whose gene coding region sequence is 1029bp (EMBL database accession number X03453) in full length, encoding a 38kDa monomeric protein consisting of 343 amino acids. Cre recombinase not only has catalytic activity, but also, similar to restriction enzymes, recognizes specific DNA sequences, i.e., loxP sites. Cre recombinase can mediate specific recombination between two LoxP sites (sequences), so that the gene sequence between the LoxP sites is deleted or recombined. Cre recombinases suitable for use herein also include mutants of Cre that retain recombinase enzyme activity. In certain embodiments, the amino acid sequence of recombinase Cre is shown in SEQ ID NO. 3 and the nucleic acid sequence is shown in SEQ ID NO. 1 at 3067-4099. Dre is a homologous recombinase similar to Cre, and similar to Cre specifically recognizes the LoxP site, dre specifically recognizes another recombination site rox. Dre recombinases suitable for use herein also include mutants of Dre that retain recombinase enzymatic activity. In certain embodiments, the amino acid sequence of recombinase Dre is shown in SEQ ID NO. 2 at 1-356. "LoxP" and "rox" as described herein have art-recognized meanings, the sequences of which are well known in the art. Illustratively, the nucleic acid sequence of LoxP is shown in SEQ ID NO. 4 and the nucleic acid sequence of rox is shown in SEQ ID NO. 5, where N represents any nucleotide.
The estrogen receptor (estrogen receptor, ER) is one of the members of the steroid hormone receptor protein superfamily. Chimeric recombinases can be produced by fusing a ligand-binding domain (LBD) of an estrogen receptor with the recombinase. The chimeric recombinase cannot enter the nucleus to bind to the recombination site and can only localize to the cytoplasm due to the presence of the estrogen receptor binding region. The chimeric recombinase can enter the nucleus to act only after the addition of estrogen. The ligand binding domain of the estrogen receptor may be mutated so that it does not bind physiological estrogens in the body, but only to exogenous inducers. The inducer forms a stable complex with the ligand binding domain of the estrogen receptor and is transported into the nucleus. The inducer includes an estrogen analog such as tamoxifen or 4-OHT. Suitable estrogen receptor ligand binding regions for use herein also include those known in the art in which the ligand binding region has been mutated, but the transmembrane domain resulting from the mutation retains the biological function of the ligand binding region (i.e., the chimeric recombinase remains capable of binding to the inducer after mutation). The mutation may be an insertion, a deletion or a substitution, and the number of amino acids to be mutated may be one or more, for example, 20 or less, preferably 10 or less, more preferably 5 or less. In some embodiments, the mutation is a substitution mutation. It is well known in the art that substitution of an amino acid with an amino acid that is chemically similar (i.e., conservative substitution) has little to no effect on the function of the resulting protein. Thus, in some preferred embodiments of the invention, the substitution is a conservative substitution. Examples of conservative substitutions include, but are not limited to, substitutions between amino acids having the same side chain group polarity, such as substitutions between nonpolar amino acids, e.g., ala, val, leu, ile, pro, phe, trp and Met, or substitutions between polar amino acids, e.g., hydrophilic amino acids Gly, ser, thr, cys, tyr, asn and gin, or substitutions between polar positively charged amino acids, e.g., lys, arg, and His, or substitutions between polar negatively charged amino acids, e.g., asp and Glu; and substitutions between fatty acid amino acids such as Ala, val, leu, ile, met, asp, glu, lys, arg, gly, ser, thr, cys, asn and Gln, etc., aromatic amino acids such as Phe and Tyr, heterocyclic amino acids such as His and Trp, etc. In an exemplary embodiment, the amino acid sequence of the estrogen receptor ligand binding region described herein is shown at positions 357-666 of SEQ ID NO. 2; the nucleic acid sequence is shown in 4153-5085 of SEQ ID NO. 1.
The polypeptides forming the fusion proteins of the invention may be linked directly or may be linked by a linker sequence. The linker may be a linker sequence capable of expressing multiple polycistronic sequences on a single vector, such as an internal ribosome entry site (internal ribosome entry site, IRES) or a 2A peptide. As is well known in the art, the 2A peptide is a short peptide that induces self-cleavage of proteins, including F2A, P2A, T A peptide and the like. The 2A peptide may also be linked to the flanking polypeptides by conventional G and S containing linkers. In one or more embodiments, the coding sequence of the P2A peptide comprises or consists of nucleotides 3001 to 3066 of SEQ ID NO. 1.
The polynucleotide sequences encoding the chimeric recombinases herein can also include one or more regulatory sequences operably linked to the chimeric recombinase. The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
Also included herein are nucleic acid molecules encoding fusion proteins of a second recombinase and an estrogen receptor ligand binding region. The second recombinant enzyme and estrogen receptor ligand binding region are defined elsewhere herein. In certain embodiments, the polynucleotide sequence of the nucleic acid molecule encodes a fusion protein Dre-ER as shown in SEQ ID NO. 2. In certain embodiments, the fusion protein of the second recombinant enzyme and the estrogen receptor ligand binding region is constitutively expressed in the host. In certain embodiments, the fusion protein of the second recombinant enzyme and the estrogen receptor ligand binding region induces expression or specific expression in the host. In certain embodiments, the mice of the invention have a coding sequence for the Dre-ER inserted at the Rosa26 gene locus.
Also included herein are nucleic acid molecules comprising the following structures: from the 5 'end to the 3' end are the recognition site for the first recombinase, the termination sequence, the recognition site for the first recombinase and the nucleic acid molecule of the marker coding sequence. Thus, expression of the marker can be regulated using a recombinase. Exemplary polynucleotide sequences for the nucleic acid molecules include the following structures: loxP-terminator sequence-LoxP-marker. In certain embodiments, the fusion protein encoded by the polynucleotide is constitutively expressed in the host. Termination sequences suitable for use herein may be any termination sequences known in the art. The label suitable for use herein may be any label known in the art. The label may be a fluorescent protein including, but not limited to, green fluorescent label (e.g., GFP, zsGreen), red fluorescent label (e.g., tdTomato, dsRed, mCherry), yellow Fluorescent Protein (YFP), cyan Fluorescent Protein (CFP), and the like. In one or more embodiments, the marker is GFP, the amino acid sequence of which is shown in SEQ ID NO. 6. In certain embodiments, the mice of the invention have a coding sequence for LoxP-stop-LoxP-GFP inserted at the Rosa26 gene locus.
Also provided herein are polynucleotide products. "Polynucleotide product" as described herein means a product comprising one or more of the polynucleotide sequences described herein. The polynucleotide sequences contained in the polynucleotide product may be the same or different. The plurality of polynucleotide sequences may be related to each other in any number or independent of each other. In one embodiment, the polynucleotide product comprises a plurality of polynucleotide sequences that are separate from one another.
In exemplary embodiments, the polynucleotide products described herein comprise: a recognition site comprising a fragment of a cell proliferation factor gene, a first recombinase coding sequence, an estrogen receptor ER coding sequence, and a second recombinase; and optionally, a polynucleotide sequence encoding a fusion protein of a second recombinant enzyme and an estrogen or a complement thereof to the receptor ER, and a polynucleotide sequence comprising the structure: from the 5 'end to the 3' end are the recognition site for the first recombinase, the termination sequence, the recognition site for the first recombinase and the polynucleotide sequence of the marker coding sequence.
Also provided herein are one or more nucleic acid constructs comprising one or more of the polynucleotide sequences described herein. The nucleic acid construct may also contain one or more regulatory sequences or sequences required for genomic homologous recombination operably linked to the sequence of the polynucleotide sequence. The nucleic acid construct may be a vector. For example, the polynucleotide sequences herein may be inserted into a recombinant expression vector or a knock-in vector. In some embodiments, the polynucleotide sequences herein are contained on the same nucleic acid construct. In certain embodiments, the polynucleotide sequences herein are contained on different nucleic acid constructs.
The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator. The polynucleotide sequences described herein are operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis via the promoter. Representative examples of these promoters are: the lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses, and some other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli. When the polynucleotides described herein are expressed in higher eukaryotic cells, transcription will be enhanced if enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene.
The vectors described herein may be transformed into suitable host cells to enable expression of the proteins described herein. In certain embodiments, the polynucleotides or cell marker systems described herein are contained in the genome of a host cell. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; filamentous fungal cells, or higher eukaryotic cells, such as mammalian cells. The host cell may also be a plant cell. Representative examples of host cells are: coli; streptomyces genus; bacterial cells of salmonella typhimurium; fungal cells such as yeast, filamentous fungi; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 The process is carried out using procedures well known in the art. Another approach is to use MgCl 2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc. The method of transfection of mouse DNA may be fertilized egg injection.
After transformation of the host cell, the resulting transformant may be cultured in a conventional manner to allow its expression of the fusion protein described herein. The medium used in the culture may be selected from various conventional media depending on the host cell used. The recombinant fusion proteins herein can be isolated and purified using various isolation methods known in the art. Such methods are well known to those skilled in the art and include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.
Thus, also included herein is a host cell comprising a protein or polynucleotide or expression vector described herein. Such host cells may constitutively express the proteins described herein, may also express the proteins described herein under certain induction conditions, and may also specifically express the proteins described herein in different host cell types. Methods of how to constitutively express, induce expression or specifically express a protein of the present invention in a host cell are well known in the art. For example, in certain embodiments, the inducible promoters are used to construct the expression vectors of the present invention to achieve inducible expression of the protein. In certain embodiments, tissue-specific expression of the protein is achieved using a tissue-specific expression promoter or associating the coding sequence of the protein with a tissue-specific gene.
Gene knock-in vectors are used to knock-in polynucleotide sequences described herein into a region of interest of the genome. Typically, the knock-in vector will contain, in addition to the polynucleotide sequence, a 5 'homology arm and a 3' homology arm required for homologous recombination of the genome. In certain embodiments, the nucleic acid constructs herein comprise a first fragment of a cell proliferation factor gene that is a 5 'homology arm, a coding sequence for a chimeric recombinase described herein, and a second fragment of a cell proliferation factor gene that is a so-called 3' homology arm. In other embodiments, the nucleic acid constructs herein contain a 5 'homology arm, a polynucleotide sequence described herein, and a 3' homology arm. When using a knock-in vector, the CRISPR/Cas9 technique can be used simultaneously to homologous recombine polynucleotide sequences to a location of interest. The CRISPR/Cas9 technology guides Cas9 nuclease to modify the genome at an insertion position by designing guide RNA for a target gene, resulting in increased homologous recombination efficiency in the modified region of the gene, and homologous recombination of a target fragment contained in a gene knock-in vector to the target site. The Cas9 nuclease may be a Cas9 nuclease as known in the art.
When the polynucleotide sequences described herein are recombined to a location of interest using CRISPR/Cas9 technology, a guide RNA target sequence for the genome of the site of interest is designed and transcribed in vitro according to the sequence to obtain a guide RNA for the gene. Meanwhile, a gene knock-in vector for recombination of a desired fragment may be constructed, the desired fragment may be a coding sequence of a protein described herein (e.g., chimeric recombinase) or a polynucleotide sequence described herein. The in vitro transcribed guide RNA and the constructed knock-in vector are then co-transferred into the cell of interest (e.g., fertilized egg), after which the cell is screened for a desired fragment knocked in at the location of interest in the genome.
The invention also includes methods of introducing (e.g., by gene recombination) the polynucleotide sequences described herein into the genome of a mouse, thereby obtaining a transgenic mouse. Methods for obtaining transgenic animals, such as transgenic mice, are known in the art, such as fertilized egg injection, embryonic stem cell injection, and the like. In addition, different transgenic mice can be bred to offspring mice having multiple polynucleotides of interest, thereby creating transgenic mouse models.
The present invention provides a method of constructing a transgenic animal comprising introducing a nucleic acid molecule, polynucleotide product or nucleic acid construct as described herein into an animal cell containing a cell proliferation factor gene, and selecting for a transgenic animal that has undergone homologous recombination.
The present invention provides a method of constructing a transgenic animal comprising the steps of: (1) Providing a first transgenic animal comprising in its genome one or more nucleic acid constructs; (2) Providing a second transgenic animal having in its genome one or more nucleic acid constructs other than the one or more nucleic acid constructs; and (3) mating the first transgenic animal and the second transgenic animal, such that homologous recombination occurs in the offspring animal to obtain the offspring transgenic animal.
The present invention provides a method of long-term cell labeling in vivo comprising providing an animal comprising a nucleic acid molecule, polynucleotide product or nucleic acid construct as described herein, and labeling cells expressing the cell proliferation factor gene in the animal in the presence of an inducer.
For example, in the co-presence of Ki67-CrexER, dreER and Rosa26-LoxP-stop-LoxP-GFP, tamoxifen induces Dre in DreER to nuclear recognize rox in the CrexER gene sequence, and homologous recombination occurs to change CrexER to Cre, such that Ki67 is co-expressed and released once Cre is expressed. Cre recognizes LoxP and activates GFP expression (fig. 1, b and fig. 3, a). The cell labeling method of the present invention can continuously track gene expression for a long period of time.
The invention also provides kits comprising the chimeric recombinases, fusion proteins, coding sequences, nucleic acid molecules, polynucleotide sequence products, nuclear constructs, or host cells described herein, as well as reagents required for knocking the nucleic acid molecules described herein into the genome of the cells.
Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, ind. Molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Examples
Example 1 construction of mice
Ki67-CrexER mice
The strategy for constructing the genetic tool mouse Ki67-CrexER (FIG. 2, a) is to knock in the 2A-CrexER2 expression frame at the termination codon site of the Mki67 gene in a site-specific manner by adopting the CRISPR/Cas9 technology through a homologous recombination mode. The brief procedure is as follows: cas9 mRNA and gRNA (SEQ ID NO: 11) are obtained by means of in vitro transcription; a homologous recombinant vector (vector) framed by the PBR322 plasmid was constructed by the method of In-Fusion cloning, and contained a 4.1kb 5 'homology arm, a 2.1kb KI fragment and a 4.0kb 3' homology arm (primer sequences used were SEQ ID NOS: 7-8). Cas9 mRNA, gRNA and donor vector were microinjected into fertilized eggs of C57BL/6J mice to obtain F0 mice. The F0 generation mouse with correct homologous recombination is obtained through long fragment PCR identification; the F0 mice were mated with C57BL/6J mice to obtain positive F1 mice.
The constructed tool mice were verified by staining with ESR (estrogen receptor immunofluorescent staining) and Ki67 or EDU (thymidine analog staining) (fig. 2, b), and the results showed that in the tissue of the small intestine, which proliferated faster, both the expression of Ki67 and ESR were concentrated in the basal acinar structures. While ESR and EdU staining results showed that both were co-localized in the same cell, indicating that the constructed tool mice proliferated with the expected consistent markers. It was further verified by mating with R26-GFP mice whether the tool mice could undergo inducible homologous recombination reactions (FIG. 2, c). The results show that the Ki67-CrexER tool mice can label cells present in pairs in the aortic endothelium of adult mice under induction of tamoxifen, indicating that the tool mice can undergo inducible homologous recombination reactions and capture the proliferative activity of the cells. The above results demonstrate successful construction of the genetic tool mouse Ki 67-CrexER.
R26-Dreer mice
An ES cell targeting mode is adopted to construct an R26-DreeR mouse, and a CAG promoter-DREERT2-polyA expression frame is inserted at a fixed point at the Rosa26 gene locus. The brief procedure is as follows: ES cell targeting vectors were constructed by the method of In-Fusion cloning, and contained a 1.087kb 5 'homology arm, CAG promoter, DREERT2 coding region, FRT-PGK-Neo-polyA-FRT, 4.259kb 3' homology arm and MC1-DTA-polyA negative selection marker (primer sequences used: SEQ ID NOS: 9-10). After linearization of the vector, the C57/129ES cells were electrotransfected. After screening by G418 and Ganc drugs, 144 resistant ES cell clones were obtained in total; and (3) carrying out long fragment PCR identification to obtain 7 positive clones of correct homologous recombination. Positive ES cell clones were amplified and injected into blasts of C57/129 mice to obtain chimeric mice. High proportion of chimeric mice were mated with C57/129 mice to obtain 4 positive F1-generation Neo-containing mice.
R26-GFP mice
R26-GFP mice were obtained from Allen institute for Brain science laboratories. The mouse has LoxP-stop-LoxP-GFP inserted into the Rosa26 gene locus.
Example 2 construction and validation of the proliferation cell marker System Protracker
Ki67-CrexER is herein incorporated; R26-Dreer; the system consisting of R26-GFP is called Protracker (FIG. 3, b). In the co-presence of Ki67-CrexER and R26-DreER, tamoxifen induces Dre in DreER to nuclear recognize rox in CrexER, and homologous recombination occurs to change CrexER to Cre such that Ki67 is released once Cre is expressed (FIG. 1, b, FIG. 3, a). Ki67-CrexER can be considered a traditional method of tracking proliferation. Adult mice obtained by mating Ki67-CrexER with R26-GFP and Protracker mice were induced with tamoxifen after adulthood, and samples were collected at the beginning of the follow-up (two days) and four weeks later, and differences in proliferation signals were observed between the two follow-up (FIGS. 3, b and c). The results showed that the traditional method of tracing proliferation did not differ from Protracker in the beginning, and that little GFP positive cell signal was detected in tissues other than the small intestine, indicating less proliferation of adult tissues or organs (FIG. 3, d). Protracker can realize long-time continuous tracking, and samples obtained after four weeks of traditional proliferation tracking and Protracker are collected, so that obvious differences between the traditional proliferation tracking method and proliferation signals tracked by Protracker are shown. In some tissue organs that proliferate relatively slowly, such as heart, lung, liver, pancreas and kidney, the ProTracker traces significantly more than the traditional proliferation tracer, indicating that the accumulation of signals due to the ProTracker system we can see almost all the cell signals that proliferate during the tracing. In muscle and brain, both systems have substantially no proliferation signal captured due to the slow proliferation of the cells themselves. The fact that the Protracker captured almost all of the small intestine epithelial cells in this faster-proliferating tissue of the small intestine suggests that the Protracker system has a higher efficiency (FIG. 3, d).
The above results demonstrate that, compared to the traditional method of tracing proliferation signals, the ProTracker restores the cell proliferation status in vivo for a certain period of time by seamlessly tracking the cell proliferation signals induced from tamoxifen for signal accumulation and finally presenting all the proliferated cell signals.
Example 3 reliability verification
R26-DreeR tool mice were mated with LoxP reporter gene mice R26-GFP mice, tamoxifen induced in adults, and individual tissues were collected for whole tissue fluorescence or slice immunofluorescence imaging (FIG. 4), which showed no GFP fluorescence signal in essentially all tissues and organs, indicating no mixed homologous recombination with LoxP for DreeR in the Protracker, a traced cell proliferation system of the invention.
ProTracker tracking in vivo proliferation signal was initiated by tamoxifen induction, to demonstrate that this whole set of system was indeed controlled by tamoxifen, we selected the littermates of the above-described conventional tracer system and the tracer proliferation tool mice of ProTracker system as controls without tamoxifen induction, and as a result found that mice without tamoxifen induction in the conventional tracer proliferation system were substantially free of green fluorescent signal (FIG. 5, a), indicating that the conventional tracer system was substantially free of leakage. The ProTracker system does not induce tamoxifen and has no green fluorescent signal in various tissues and organs of the whole body, which indicates that the ProTracker system is truly regulated by tamoxifen induction. This shows that the proliferating cell labeling and tracing system of the present invention can truly and reliably track in vivo cell proliferation signals.
Example 4 tracking of proliferating cells of liver Using Protracker
The Protracker system constructed in example 2 was used to specifically study cells that proliferated in vivo. The liver is an organ with regeneration capability consisting of a plurality of liver lobules, wherein liver cells have a certain proliferation capability. Liver as a metabolic organ simultaneously performs different metabolic functions in different areas of the liver lobule, and liver cells along the blood flow from portal vein to central vein in the liver lobule structure constituting the liver can be roughly divided into three areas: E-CAD+ region 1, GS+ region 3, and region 2 located therebetween (FIG. 6, b). Since cells in different regions perform different metabolic functions, it is also considered in the art that these cells have different proliferation capacities. Previous studies have used molecular markers of hepatocytes in different regions to construct genetic tools mice to lineage trace hepatocytes in these regions, and it was concluded that hepatocytes in the central venous region have greater proliferative capacity in physiological homeostasis. However, the lineage tracing technology using the specific area hepatocyte markers can only trace the change of a single population of hepatocytes each time, and cannot explore the proliferation change of hepatocytes at the whole level. To study the proliferation of hepatocytes from an overall level, a set of genetic lineage tracing techniques independent of molecular markers in specific regions of hepatocytes was used. The Protracker system of the present invention can meet this requirement, and study the source and fate of newly generated hepatocytes.
The ProTracker system mice were adult and then induced with tamoxifen to initiate cell proliferation tracking, and samples were taken at different time points after induction (FIG. 6, a), and an analysis was made as to where hepatocytes initially began to proliferate and where these proliferated cells then migrated. The results showed that at the very beginning of the trace (zero and two days after tamoxifen induction), essentially no proliferative hepatocyte signals were captured (fig. 6, c). When the tracking time was prolonged to two weeks, sporadic proliferation signals appeared at the 1-region and 2-region positions of the hepatic lobules, while no proliferation signals appeared at the 3-region. By the fourth week, the proliferation signal of region 2 increased significantly, as did the proliferation signal of region 1, while region 3 still showed substantially no proliferation. Following this, the cell proliferation was maintained at weeks 6, 8, 10, and 12, with zone 2 proliferation occurring at most 1 and less cell proliferation signal occurring in zone 3 (FIGS. 6, d and e). Therefore we consider that the most vigorous hepatocyte proliferation in the hepatic lobules of the liver at physiological homeostasis is the region 2 hepatocytes located intermediate the portal vein and central vein regions.
Example 5 tracking of proliferating cells of the heart Using Protracker
Cardiomyocytes were initially considered incapable of proliferation as a terminally differentiated cell type. Recent studies suggest that cardiomyocytes in adult hearts can produce new cardiomyocytes by proliferation. However, the studies mainly utilize the isotope incorporation method, which introduces the problem of difficult detection on the one hand, and on the other hand, since the cardiomyocytes are polyploid and the isotope incorporation mainly detects the nuclei, it is difficult to distinguish between the nuclear polyploidization phenomenon occurring in the cardiomyocytes and the cell division phenomenon. In addition, whether newly generated cardiomyocytes are regional or not and whether generation of new cardiomyocytes by cell division into two can be detected remains a problem to be solved.
Cardiomyocyte proliferation was relatively slow, and potential proliferation signals were not captured using conventional proliferation marker staining and DNA analogue incorporation. While traditional lineage tracing techniques also miss most of the proliferated cardiomyocytes because of the short window of tamoxifen action, the pro tracker system of the present invention allows us to continuously capture the active signal of Ki67 (fig. 7, b) because DreER enters the nucleus under tamoxifen action to change Ki67-CrexER to Ki67-Cre, thus allowing the signal to be superimposed from the very beginning of signal capture until the detection time (fig. 7,a). The superimposed proliferation signals eventually appear in the larger volume of cells, cardiac cells, also facilitating the observation of the location as well as the distance between all signals as a whole.
Tamoxifen induction was performed on adult ProTracker mice, and heart tissue was harvested three months later and immunofluorescent stained to find that many gfp+ cardiomyocytes were present in the heart. Results of immunofluorescent staining were statistically found that approximately 0.7% ± 0.14% of cardiomyocytes developed active expression of Ki67 within three months. Furthermore, we found captured GFP + Most of the cardiomyocytes in the ventricular septum are located on the left ventricular wall near the endocardial side, while the cardiomyocytes in the ventricular septum are also significantly more gfp+ cardiomyocytes on the side facing the left ventricular chamber than on the side facing the right ventricular chamber. The right ventricle wall of the heart was substantially free of gfp+ cardiomyocytes. To this end, we have found a population of actively proliferating cardiomyocytes in the left ventricular chamber of the annulus in adult hearts using the ProTracker system.
Actively proliferating cells expressed Ki67 in all G1, S, G, M phases of the cell cycle, so gfp+ proliferation signals captured by protacker included cell division as well as nuclear division (fig. 8, a). Next, further studies were performed on captured GFP+ cardiomyocytes. The Hoechst staining of isolated cardiomyocytes revealed that GFP+ cardiomyocytes contained single-, double-and polynuclear individuals (FIG. 8, b), and the statistics of the number of nuclei of these isolated cardiomyocytes Now GFP + The increased number of single nuclei, decreased number of double nuclei and multiple nuclei of the cardiomyocytes compared to GFP-cardiomyocytes indicates the occurrence of mitosis in the cardiomyocytes. Sequential multi-slice confocal scans captured individual cardiomyocytes and two immediately adjacent cardiomyocytes (FIGS. 8,c and e). Successive layer scans show that the individual cardiomyocytes captured are multinucleated and that the two immediately adjacent cardiomyocytes are single-cored myocytes (fig. 8, d and f). If the captured immediately adjacent cardiomyocytes were considered as a result of cell division, actively proliferating cells (GFP + About 8% of the cardiomyocytes) involved in cell division. To this end, we studied the proliferation of adult hearts using the Protracker system and found that a population of proliferating cardiomyocytes located in the left ventricular chamber of the annulus also captured the phenomenon of cardiomyocyte division in adult hearts.
The invention realizes the aim of seamlessly tracking in vivo cell proliferation signals by designing a novel genetic tool mouse, and researches the proliferation of liver cells and myocardial cells in the adult mouse. Proliferation of various types of cells in the body can also be tracked using this technique. In addition, by replacing different DreER tool mice hybridized, the proliferation of cells of a certain group can be tracked independently in vivo, and the method has important significance for understanding the dynamic change of cells of various groups in vivo.
After reading the above teachings of the present invention, a person skilled in the art may make various changes or modifications to the present invention, these equivalents likewise falling within the scope of the invention as defined by the writing of the claims appended hereto.
Sequence listing
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taaaatagca gaatgagttg gctcagcagt tgagagcacc aaatactctt tcaaaggagc 420
cagttgttat tcctaggact cacatggctc acaactatct gtaactccag ttccagggga 480
tttgatggtc tcttctgacc tccttaaaca ccaagcaggc atatgcatag gtgaaggcaa 540
aaaacataaa aattaaaagg aataaatatg atttaaaaaa aataaataaa aaggcagaat 600
aagcccagta ttgtctatct cctaagcaaa taagtgaaaa taggtaaatt ctcctcagtg 660
ggaagtgtgt tgtttcagca tcccaagctc acagtactaa gctagtagac tgtaaaccca 720
tgtgctgtcc gtgctttgca tcattcccag tggcgtgctt gcattaagca gtagtctagg 780
accaggtaaa gatggggtgg gcaggagtga cacataattg ttctggacat tcaagttgaa 840
cctgaacata ctctgaacat tctgaaacag ctgatgggag ataaaggtat tgacagccgc 900
tcgaggcaga ggtgtgctga gctggcagtc ctacatgtga ctggcacagc acaagaagac 960
ccaggctttc cagaggtcat gaaacatccc atatagctga gggatggagc aagaggcatg 1020
gaagtctagt gcgaaggtgc agctaaagca cccagaatgg cggctctcaa cctcccaatg 1080
ctgtgactct ttaatacagt tcttctgtgg tgatccccaa ccataaaatt attttattgc 1140
tacttgatca ctaatgttgc tactgttatg aatcataatg taaatatctg gtatgcatga 1200
tatctgatat atgaaacaac ctgtgaaagg gttgtttgac ccccacaaag ggcttgagaa 1260
ccatggaagc gaggctcagg aaaggagagc aatacccagg aagtggaggt ggctgtccgg 1320
aagcagaaac tatctcatgg tggcaagcct tcattacaga caccaagaca caccaggaac 1380
tcagcctcat aggcttaaca agtatcttat ctttcctcag agctctaagc acagcttcat 1440
caccttgaaa gtagtacttt atcagaagga aatagaagga ataaaaccca ggttttttta 1500
gtcaaatgat cctgaacaca acaggcaagg cctgagggtg atcaggccag gtcatcgtgt 1560
catagacact caggtctctt ctcctcactg gtgcccagcg gatgtcatac actgacgagt 1620
tttcccagga ctatatcttt cttgtctgtt ctgttgtcca taggcccaga ttctacatat 1680
gtgtgtgtgg gaggggtggg tgcttcctgg cggtccatcc tgcaagtatg ctggagaagc 1740
aagcctctta tctggtgtgt gtgcctttct aacatgtgta cagtagatcc atctacctgt 1800
tattttctag aattcaacag cattcacata ccaagatctt cttgtccata ctgagcctca 1860
cacttaagag ttcctgtttt ccgtctccct ttcttaactg tccataatca ctcataaaac 1920
tgtgtctaaa gtatgcccag catcaccctc ggctttttct aatttttgtt ggatgggcct 1980
tgtgtttatc aggtaccaga actttgggtc atttgctcta agaggctatt gtgacctttt 2040
gctttctgta gattggatcc ccgcttccag gtagatgggc ctgcctctat ctccccactg 2100
ccttagagga cccactatcc ttttgcagcc acataggaga cctcaggaca cagtgactgt 2160
cctttgtctg tgggaagttg gctttaggat acttaagttt tcatctaggc cacagtcaaa 2220
ttttgtgaat gatgtttttt aattagtgaa ccacatacag tgatagagac cgtgtatgct 2280
ttagaaactt gtgaaagagc acagagtttg agttttaaaa actaagttaa aaaaaaacat 2340
ttaggaagaa acaaccttat ggtaagcatt gtaaaaggat ttccaacttt aatttttttc 2400
tttttaaaaa cactttgtag ccaggcagtg gtggtgcaca cctttaatcc cagcacttgg 2460
gaggcagagg caggtggatt tctgagttcg aggccagcct ggcctacaga gtgagttcca 2520
ggacagccag ggctatacag aggaaccctg tcttgataaa ccaaacaaac aaaaaagaaa 2580
aacactttgt ttttgttttg tgggtttttt tttttaatgt atactgagta ttttgccgtg 2640
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tatcatgtgc ttgcctgcgg 2700
aggtcagtca gaaaagggca tctggttccc tggatggttg tgagccacca tgtggatgct 2760
gggagttaaa cttaggtcct ctgtaagtgc agaaagtgcc cctagacact gagccatctt 2820
tccagacctt caatgttaat ttatagatga gagacctgaa tgacacctag taaggacaag 2880
gggctcattg agttgaggtg atcacaaaaa ttgttccttc atattattta ggagtgtggt 2940
tttttttttc ccaaacagga tgaagacatt gtatgcacca agaagttaag aacaagaagt 3000
ggaagcggag ctactaactt cagcctgctg aagcaggctg gcgacgtgga ggagaaccct 3060
ggtcctatgg gctccaattt actgaccgta caccaaaatt tgcctgcatt accggtcgat 3120
gcaacgagtg atgaggttcg caagaacctg atggacatgt tcagggatcg ccaggcgttt 3180
tctgagcata cctggaaaat gcttctgtcc gtttgccggt cgtgggcggc atggtgcaag 3240
ttgaataacc ggaaatggtt tcccgcagaa cctgaagatg ttcgcgatta tcttctatat 3300
cttcaggcgc gcggtctggc agtaaaaact atccagcaac atttgggcca gctaaacatg 3360
cttcatcgtc ggtccgggct gccacgacca agtgacagca atgctgtttc actggttatg 3420
cggcggatcc gaaaagaaaa cgttgatgcc ggtgaacgtg caaaacaggc tctagcgttc 3480
gaacgcactg atttcgacca ggttcgttca ctcatggaaa atagcgatcg ctgccaggat 3540
atacgtaatc tggcatttct ggggattgct tataacaccc tgttacgtat agccgaaatt 3600
gccaggatca gggttaaaga tatctcacgt actgacggtg ggagaatgtt aatccatatt 3660
ggcagaacga aaacgctggt tagcaccgca ggtgtagaga aggcacttag cctgggggta 3720
actaaactgg tcgagcgatg gatttccgtc tctggtgtag ctgatgatcc gaataactac 3780
ctgttttgcc gggtcagaaa aaatggtgtt gccgcgccat ctgccaccag ccagctatca 3840
actcgcgccc tggaagggat ttttgaagca actcatcgat tgatttacgg cgctaaggat 3900
gactctggtc agagatacct ggcctggtct ggacacagtg cccgtgtcgg agccgcgcga 3960
gatatggccc gcgctggagt ttcaataccg gagatcatgc aagctggtgg ctggaccaat 4020
gtaaatattg tcatgaacta tatccgtaac ctggatagtg aaacaggggc aatggtgcgc 4080
ctgctggaag atggcgatct aactttaaat aattggcatt atttaaagtt actcgagcca 4140
tctgctggag acatgagagc tgccaacctt tggccaagcc cgctcatgat caaacgctct 4200
aagaagaaca gcctggcctt gtccctgacg gccgaccaga tggtcagtgc cttgttggat 4260
gctgagcccc ccatactcta ttccgagtat gatcctacca gacccttcag tgaagcttcg 4320
atgatgggct tactgaccaa cctggcagac agggagctgg ttcacatgat caactgggcg 4380
aagagggtgc caggctttgt ggatttgacc ctccatgatc aggtccacct tctagaatgt 4440
gcctggctag agatcctgat gattggtctc gtctggcgct ccatggagca cccagtgaag 4500
ctactgtttg ctcctaactt gctcttggac aggaaccagg gaaaatgtgt agagggcatg 4560
gtggagatct tcgacatgct gctggctaca tcatctcggt tccgcatgat gaatctgcag 4620
ggagaggagt ttgtgtgcct caaatctatt attttgctta attctggagt gtacacattt 4680
ctgtccagca ccctgaagtc tctggaagag aaggaccata tccaccgagt cctggacaag 4740
atcacagaca ctttgatcca cctgatggcc aaggcaggcc tgaccctgca gcagcagcac 4800
cagcggctgg cccagctcct cctcatcctc tcccacatca ggcacatgag taacaaaggc 4860
atggagcatc tgtacagcat gaagtgcaag aacgtggtgc ccctctatga cctgctgctg 4920
gaggcggcgg acgcccaccg cctacatgcg cccactagcc gtggaggggc atccgtggag 4980
gagacggacc aaagccactt ggccactgcg ggctctactt catcgcattc cttgcaaaag 5040
tattacatca cgggggaggc agagggtttc cctgccacag cttgataact aactttaaat 5100
aattggcatt atttaaagtt atgataagaa caagaagtta ccagaaaagt gaaactatgt 5160
agcaaagaca tttaagaagg aaaagtaaat ttgacttagt gataagttcc agtgtggttt 5220
tcacctccag tgtaaagatg aactgtaaat actactgcta ctgcctgagt ttaaggaagg 5280
aagctttgag ctttcctggt catactctct tcagacgcca atggaggtca tgaggaagat 5340
caccagggat ctcagcgcaa ttacagttta ggggtgagca ggcagaaatg tggccctctg 5400
tcctatccaa taaagctctg aaattcgctg cctcctttgg cctctctgac aactgcagct 5460
gctcccctct gccctcatga aggaggggaa ggtggtgccc ctccattcat tagacattgg 5520
ttgtgcagtt atatcagcca accttacaca ggatgactgt acggtggagt ggttggtttg 5580
taggctacac cattagtcac ttacgcaagt cagcctaatc ctctgggcct gtgacctttg 5640
ggagaaacat ctgacaagga tggctgccga gctcccttca ggggcacggg tcgctatgtt 5700
aaagagcggt tgatgtctgt gcttttcatt aggcctctgt attgagtgga ttggctgcct 5760
tgcctgtgga acctttgctg ctggggagtc tcctgtcccc actggagtct ccactccagt 5820
ctcctgtcct agcgtctgct tttatcacgg gctttctctg acctcttgcc tggcagcaca 5880
aggccatcct ggtgtctggt atgagatgct tatcttccaa gtttcacttt aaccctaaac 5940
tcttttctgt tggaaaccac tgcgcatttg catatgcaac tttgtgcttt tcactctgcc 6000
tgctagtccc ctttctgttt tccagcagta acatatctgc tggtgctgga agagagccta 6060
gagtgtgccc tggtcagcca ttgccctaac ctcttcactt ctccatctcc tgtctgagat 6120
acaggtgaag aacactgggt acgcaggtga gaaacactga gtggaggccg ggatttagca 6180
ttttgggtga gtctgggagt tctgccattt catctacctc aggaattctg taatcaagga 6240
atggcaactg gttattaata agggggcaaa agcttcatag ggtgggtaac agtggaactg 6300
gcaaaggaga ttgtgtagag cagaaggcac aggaaaagag cgcccttttt acctgttaga 6360
gggtgtgagg catgaaagtg cccttaattg acttaaatcc taaagtcaaa gtctttgaag 6420
taacaggaac cttgactatg aattgctctg atgtagaatt agaaatatca catgtatgtg 6480
ggaaattgta gtcaactgca tgctgattga atggaactgg gtgataaggg aaaggcctgc 6540
tcagttatag gaaattctgt ctgagccatg ttagcacatt ttctcactta ggacagatgt 6600
gacggctctg aagcagctgc tatgcaggca agaaggcaag agcagattag cagaacctat 6660
gtctgagctg ggcctggtga cataggtctg caaccccagc attgggatat ggagtcaggt 6720
atatgagtgc ccgaggcttg ctagccagcc accctagcca aagaggatcc agtagaaaga 6780
tgtcccaaat cagcctacat acatgtctgc ttgtgtgggc tgatgtgtgc acttggtatg 6840
tatatatgca cacacatgca gccaccatgt aacctaaaac gctcatttga gggtgatacc 6900
attgccaaga cattcttaga acacatcctc tatttatctc tgtgtgcaca tctgagaaag 6960
acccacttgt tggttgattg taacaaatat ccacccattc ctcaagtgtt tagctatggt 7020
ccctagcaat gtcagtttcc cagcagaaag catgatggga gattcccaag aaaggagtgc 7080
tgtacttttt gcctcccaga tctgtgactc ttcctgtttt gttgtcattt gctcctgccc 7140
ttctcataaa cagctactgt tttccctgcc tggaacttga cccagccccg cacttcatca 7200
attgtattca ttggaatgat gaacttagct ccaagaagct tcctggcctc tccactgcag 7260
ccactgtccc gggttaggaa cggcaggtcc ttagttgtca gcagcatcta ggcacctagt 7320
gagaatcggc atctgtatta gtcagggttc tctagagtca cagaacttag gaatagtctc 7380
tatatagtaa aggaatttat tgatgattta tagtagtcca attcccaaca atggttcagt 7440
agaagctgtg aatggaagtc caaagatcta gcagttactc agtcccacac ggcaagcagg 7500
cgaaggagca agagccagac tcccttcttc caatgtcctt atatggtctc cagcagaagg 7560
tgtagcccag attaaaggtc tgtcccacca cacctttaat cccagatgac cttgaactca 7620
gagatctccc tgtcttaatc ttctggaatc catagccact atgcctcaag atctccatac 7680
caagatccag atcagaaact tccatctccc agcctccaga ttagggtcac tggtgagcct 7740
tccaattctg gattgtagtt cattccaaat atagtcaagt tgacagctgg gaatagccac 7800
tacagcatcc taaatgcaat tttcatcccc ttgactaaac tgatttagtt taatagcatg 7860
taatctcagc ttgcctgatg attgcaatgt gacttggggc aaatctttaa caggcagttt 7920
tctggtctat agaatgatgt tctcagtgct ccatctcagg gtagttaaga tgaacagaat 7980
agaatactgc ttgcagctcc tgtagccttt ggccagtgct tggagtcaag ctgggtcatg 8040
agggctttct ccactgagaa ggtagaagga agatttggag caccgaagtc tcagcactag 8100
attttatatg atgtcctgaa cagggaa 8127
<210> 2
<211> 666
<212> PRT
<213> Artificial Sequence
<400> 2
Met Gly Ala Ser Glu Leu Ile Ile Ser Gly Ser Ser Gly Gly Phe Leu
1 5 10 15
Arg Asn Ile Gly Lys Glu Tyr Gln Glu Ala Ala Glu Asn Phe Met Arg
20 25 30
Phe Met Asn Asp Gln Gly Ala Tyr Ala Pro Asn Thr Leu Arg Asp Leu
35 40 45
Arg Leu Val Phe His Ser Trp Ala Arg Trp Cys His Ala Arg Gln Leu
50 55 60
Ala Trp Phe Pro Ile Ser Pro Glu Met Ala Arg Glu Tyr Phe Leu Gln
65 70 75 80
Leu His Asp Ala Asp Leu Ala Ser Thr Thr Ile Asp Lys His Tyr Ala
85 90 95
Met Leu Asn Met Leu Leu Ser His Cys Gly Leu Pro Pro Leu Ser Asp
100 105 110
Asp Lys Ser Val Ser Leu Ala Met Arg Arg Ile Arg Arg Glu Ala Ala
115 120 125
Thr Glu Lys Gly Glu Arg Thr Gly Gln Ala Ile Pro Leu Arg Trp Asp
130 135 140
Asp Leu Lys Leu Leu Asp Val Leu Leu Ser Arg Ser Glu Arg Leu Val
145 150 155 160
Asp Leu Arg Asn Arg Ala Phe Leu Phe Val Ala Tyr Asn Thr Leu Met
165 170 175
Arg Met Ser Glu Ile Ser Arg Ile Arg Val Gly Asp Leu Asp Gln Thr
180 185 190
Gly Asp Thr Val Thr Leu His Ile Ser His Thr Lys Thr Ile Thr Thr
195 200 205
Ala Ala Gly Leu Asp Lys Val Leu Ser Arg Arg Thr Thr Ala Val Leu
210 215 220
Asn Asp Trp Leu Asp Val Ser Gly Leu Arg Glu His Pro Asp Ala Val
225 230 235 240
Leu Phe Pro Pro Ile His Arg Ser Asn Lys Ala Arg Ile Thr Thr Thr
245 250 255
Pro Leu Thr Ala Pro Ala Met Glu Lys Ile Phe Ser Asp Ala Trp Val
260 265 270
Leu Leu Asn Lys Arg Asp Ala Thr Pro Asn Lys Gly Arg Tyr Arg Thr
275 280 285
Trp Thr Gly His Ser Ala Arg Val Gly Ala Ala Ile Asp Met Ala Glu
290 295 300
Lys Gln Val Ser Met Val Glu Ile Met Gln Glu Gly Thr Trp Lys Lys
305 310 315 320
Pro Glu Thr Leu Met Arg Tyr Leu Arg Arg Gly Gly Val Ser Val Gly
325 330 335
Ala Asn Ser Arg Leu Met Asp Ser Ala Ser Gly Ala Arg Arg Ile Cys
340 345 350
Val Arg Gly Ser Met Arg Ala Ala Asn Leu Trp Pro Ser Pro Leu Met
355 360 365
Ile Lys Arg Ser Lys Lys Asn Ser Leu Ala Leu Ser Leu Thr Ala Asp
370 375 380
Gln Met Val Ser Ala Leu Leu Asp Ala Glu Pro Pro Ile Leu Tyr Ser
385 390 395 400
Glu Tyr Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu
405 410 415
Leu Thr Asn Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp Ala
420 425 430
Lys Arg Val Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln Val His
435 440 445
Leu Leu Glu Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val Trp
450 455 460
Arg Ser Met Glu His Pro Val Lys Leu Leu Phe Ala Pro Asn Leu Leu
465 470 475 480
Leu Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile Phe
485 490 495
Asp Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu Gln
500 505 510
Gly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser Gly
515 520 525
Val Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys Asp
530 535 540
His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His Leu
545 550 555 560
Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu Ala
565 570 575
Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser Asn Lys Gly
580 585 590
Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn Val Val Pro Leu Tyr
595 600 605
Asp Leu Leu Leu Glu Ala Ala Asp Ala His Arg Leu His Ala Pro Thr
610 615 620
Ser Arg Gly Gly Ala Ser Val Glu Glu Thr Asp Gln Ser His Leu Ala
625 630 635 640
Thr Ala Gly Ser Thr Ser Ser His Ser Leu Gln Lys Tyr Tyr Ile Thr
645 650 655
Gly Glu Ala Glu Gly Phe Pro Ala Thr Ala
660 665
<210> 3
<211> 344
<212> PRT
<213> Artificial Sequence
<400> 3
Met Gly Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro
1 5 10 15
Val Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe
20 25 30
Arg Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser
35 40 45
Val Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp
50 55 60
Phe Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln
65 70 75 80
Ala Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu
85 90 95
Asn Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn
100 105 110
Ala Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala
115 120 125
Gly Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp
130 135 140
Gln Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg
145 150 155 160
Asn Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala
165 170 175
Glu Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly
180 185 190
Arg Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala
195 200 205
Gly Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg
210 215 220
Trp Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe
225 230 235 240
Cys Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln
245 250 255
Leu Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu
260 265 270
Ile Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser
275 280 285
Gly His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly
290 295 300
Val Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn
305 310 315 320
Ile Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met
325 330 335
Val Arg Leu Leu Glu Asp Gly Asp
340
<210> 4
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 4
ataacttcgt atannntann ntatacgaag ttat 34
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 5
taactttaaa taatnnnnat tatttaaagt ta 32
<210> 6
<211> 765
<212> PRT
<213> Artificial Sequence
<400> 6
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Phe Thr Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Lys Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Glu
225 230 235 240
Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly
245 250 255
Pro Ala Pro Gly Ser Met Ser Gly Gly Glu Glu Leu Phe Ala Gly Ile
260 265 270
Val Pro Val Leu Ile Glu Leu Asp Gly Asp Val His Gly His Lys Phe
275 280 285
Ser Val Arg Gly Glu Gly Glu Gly Asp Ala Asp Tyr Gly Lys Leu Glu
290 295 300
Ile Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr
305 310 315 320
Leu Val Thr Thr Leu Cys Tyr Gly Ile Gln Cys Phe Ala Arg Tyr Pro
325 330 335
Glu His Met Lys Met Asn Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
340 345 350
Tyr Ile Gln Glu Arg Thr Ile Gln Phe Gln Asp Asp Gly Lys Tyr Lys
355 360 365
Thr Arg Gly Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile
370 375 380
Glu Leu Lys Gly Lys Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His
385 390 395 400
Lys Leu Glu Tyr Ser Phe Asn Ser His Asn Val Tyr Ile Arg Pro Asp
405 410 415
Lys Ala Asn Asn Gly Leu Glu Ala Asn Phe Lys Thr Arg His Asn Ile
420 425 430
Glu Gly Gly Gly Val Gln Leu Ala Asp His Tyr Gln Thr Asn Val Pro
435 440 445
Leu Gly Asp Gly Pro Val Leu Ile Pro Ile Asn His Tyr Leu Ser Thr
450 455 460
Gln Thr Lys Ile Ser Lys Asp Arg Asn Glu Ala Arg Asp His Met Val
465 470 475 480
Leu Leu Glu Ser Phe Ser Ala Cys Cys His Thr His Gly Met Asp Glu
485 490 495
Leu Tyr Arg Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser
500 505 510
Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val
515 520 525
Ser Lys Gln Ile Leu Lys Asn Thr Gly Leu Gln Glu Ile Met Ser Phe
530 535 540
Lys Val Asn Leu Glu Gly Val Val Asn Asn His Val Phe Thr Met Glu
545 550 555 560
Gly Cys Gly Lys Gly Asn Ile Leu Phe Gly Asn Gln Leu Val Gln Ile
565 570 575
Arg Val Thr Lys Gly Ala Pro Leu Pro Phe Ala Phe Asp Ile Leu Ser
580 585 590
Pro Ala Phe Gln Tyr Gly Asn Arg Thr Phe Thr Lys Tyr Pro Glu Asp
595 600 605
Ile Ser Asp Phe Phe Ile Gln Ser Phe Pro Ala Gly Phe Val Tyr Glu
610 615 620
Arg Thr Leu Arg Tyr Glu Asp Gly Gly Leu Val Glu Ile Arg Ser Asp
625 630 635 640
Ile Asn Leu Ile Glu Glu Met Phe Val Tyr Arg Val Glu Tyr Lys Gly
645 650 655
Arg Asn Phe Pro Asn Asp Gly Pro Val Met Lys Lys Thr Ile Thr Gly
660 665 670
Leu Gln Pro Ser Phe Glu Val Val Tyr Met Asn Asp Gly Val Leu Val
675 680 685
Gly Gln Val Ile Leu Val Tyr Arg Leu Asn Ser Gly Lys Phe Tyr Ser
690 695 700
Cys His Met Arg Thr Leu Met Lys Ser Lys Gly Val Val Lys Asp Phe
705 710 715 720
Pro Glu Tyr His Phe Ile Gln His Arg Leu Glu Lys Thr Tyr Val Glu
725 730 735
Asp Gly Gly Phe Val Glu Gln His Glu Thr Ala Ile Ala Gln Leu Thr
740 745 750
Ser Leu Gly Lys Pro Leu Gly Ser Leu His Glu Trp Val
755 760 765
<210> 7
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 7
tggattttgc tgcagttatt tgtgtatag 29
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 8
cacataaatt aaacatgact tggttac 27
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 9
tccgagcgtg gtggagccgt tct 23
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 10
tactaccttg ttctgataga aatattt 27
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 11
ataaattaac attgaaggtc 20

Claims (15)

1. A recombinase system, characterized in that,
the system comprises
(1) A nucleic acid molecule comprising, in order from the 5 'end to the 3' end, a 5 'homology arm, a coding sequence for a first recombinase, a recognition site for a second recombinase, an estrogen receptor ER coding sequence, a recognition site for a second recombinase, and a 3' homology arm, said 5 'homology arm and 3' homology arm being capable of recombining sequences therebetween into a genome such that the sequences therebetween are co-expressed with a cytokine gene in the genome, and
(2) A nucleic acid molecule encoding a fusion protein comprising, in order from the N-terminus to the C-terminus, a second recombinant enzyme and an estrogen receptor ER,
(3) A nucleic acid molecule comprising the structure: from the 5 'end to the 3' end are, respectively, a recognition site for the first recombinase, a termination sequence, a recognition site for the first recombinase and a marker coding sequence, or
The system comprises
(a) A first nucleic acid construct comprising a nucleic acid molecule comprising, in order from the 5 'end to the 3' end, a 5 'homology arm, a coding sequence for a first recombinase, a recognition site for a second recombinase, an estrogen receptor ER coding sequence, a recognition site for a second recombinase, and a 3' homology arm, the 5 'homology arm and the 3' homology arm being capable of recombining sequences therebetween into a genome such that the sequences therebetween are co-expressed with a cytokine gene in the genome, and
(b) A second nucleic acid construct having a polynucleotide sequence encoding a fusion protein comprising a second recombinant enzyme and an estrogen receptor ER in sequence from the N-terminus to the C-terminus,
(c) A third nucleic acid construct having a polynucleotide sequence comprising the structure: from the 5 'end to the 3' end are the recognition site of the first recombinase, a termination sequence, the recognition site of the first recombinase and a marker coding sequence, respectively,
the first recombinase and the second recombinase are Cre and Dre or Dre and Cre respectively, wherein the recognition site of Cre is LoxP and the recognition site of Dre is rox.
2. The recombinase system of claim 1 wherein the 5 'homology arms and 3' homology arms of (1) and (a) are capable of recombining sequences therebetween to the 5 'or 3' end of the cell proliferation factor gene, and wherein the nucleic acid molecule of (1) or (a) has one or more features selected from the group consisting of:
The cell proliferation factor is Ki67 or PCNA,
the nucleic acid sequence of the 5' homology arm is shown as 1 st-3000 th nucleotide of SEQ ID NO. 1,
the nucleic acid sequence of the 3' homology arm is shown as 5128-8127 nucleotide of SEQ ID NO. 1,
the amino acid sequence of Dre is shown as amino acids 1-356 of SEQ ID NO. 2,
the amino acid sequence of Cre is shown as SEQ ID NO. 3,
the nucleic acid sequence of LoxP is shown as SEQ ID NO. 4,
the nucleic acid sequence of rox is shown as SEQ ID NO. 5,
the amino acid sequence of the estrogen receptor ER is shown as 357-666 amino acids of SEQ ID NO. 2.
3. A host cell comprising the system of claim 1 or 2.
4. A method of constructing a transgenic animal comprising:
three kinds of F 0 Mating any two of the animals of the generation and then carrying out homologous recombination F 1 Substituted animals and third F 0 Mating of animals, at F 2 Homologous recombination occurs in the animal of the generation to obtain a transgenic animal comprising the first, second and third polynucleotide sequences,
wherein,,
F 0 the genome of the first animal comprises a first polynucleotide sequence which comprises a 5 'homologous arm, a first recombinase coding sequence, a recognition site of a second recombinase, an estrogen receptor ER coding sequence, a recognition site of the second recombinase and a 3' homologous arm from the 5 'end to the 3' end in sequence, and is co-expressed with a cell proliferation factor gene in the genome;
F 0 The genome of the second animal comprises a second polynucleotide sequence encoding a fusion protein comprising a second recombinant enzyme and an estrogen receptor ER in sequence from the N-terminus to the C-terminus,
F 0 the genome of the third animal contains a third polynucleotide sequence which is a recognition site of the first recombinase, a termination sequence, a recognition site of the first recombinase and a marker coding sequence in sequence from the 5 'end to the 3' end.
5. The method of claim 4, wherein the animal is a mouse.
6. The method of claim 4, wherein the first and second recombinant enzymes, estrogen receptor ER and cell proliferation factor are as set forth in claim 1 or 2.
7. A method of constructing a transgenic animal comprising introducing into animal cells and culturing any one, two or three of a first, second, and third polynucleotide sequence, and selecting a transgenic animal comprising the first, second, and third polynucleotide sequences in its genome, wherein
The first polynucleotide sequence is a 5 'homology arm, a first recombinase coding sequence, a recognition site for a second recombinase, an estrogen receptor ER coding sequence, a recognition site for a second recombinase and a 3' homology arm in sequence from the 5 'end to the 3' end, and the first polynucleotide sequence is co-expressed with a cell proliferation factor gene in a genome in the transgenic animal,
The second polynucleotide sequence encodes a fusion protein comprising a second recombinant enzyme and an estrogen receptor ER in sequence from the N-terminus to the C-terminus,
the third polynucleotide sequence is a recognition site of the first recombinase, a termination sequence, a recognition site of the first recombinase and a marker coding sequence in sequence from the 5 'end to the 3' end.
8. The method of claim 7, wherein the cell is an animal ES cell.
9. The method of claim 7, wherein the animal is a mouse.
10. The method of claim 7, wherein the first and second recombinant enzymes, estrogen receptor ER and cell proliferation factor are as set forth in claim 1 or 2.
11. A method of long-term cell labelling in vivo comprising labelling cells expressing a cell proliferation factor gene in vivo in an animal comprising first, second and third polynucleotide sequences in the presence of an inducer of interaction with an estrogen receptor ER, wherein,
the first polynucleotide sequence comprises a 5 'homology arm, a first recombinase coding sequence, a recognition site of a second recombinase, an estrogen receptor ER coding sequence, a recognition site of the second recombinase and a 3' homology arm from the 5 'end to the 3' end in sequence, and the first polynucleotide sequence is co-expressed with a cell proliferation factor gene in a genome in a transgenic animal,
The second polynucleotide sequence encodes a fusion protein comprising, in order from the N-terminus to the C-terminus, a second recombinant enzyme and an estrogen receptor ER, and
the third polynucleotide sequence is a recognition site, a termination sequence, a first recombination site and a marker coding sequence of the first recombinase in sequence from the 5 'end to the 3' end.
12. The method of claim 11, wherein the animal is a mouse.
13. The method of claim 11, wherein the first and second recombinant enzymes, estrogen receptor ER and cell proliferation factor are as set forth in claim 1 or 2.
14. Use of the system of claim 1 or 2 or the host cell of claim 3 in long-term cell labeling or cell tracing.
15. A kit comprising the system of claim 1 or 2 or the host cell of claim 3, and reagents required to knock a nucleic acid molecule into the genome of the cell.
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