CN114921480A - Construction method of bone cell Terc gene knockout mouse model - Google Patents
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- C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
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Abstract
The invention discloses a method for constructing a bone cell Terc gene knockout mouse model, which comprises the following steps: performing flox modification on the Terc gene, and respectively inserting a loxp sequence (SEQ ID NO.:1) at the upstream and the downstream of the Terc gene; inserting a terminator sequence (SEQ ID NO: 2) between the Terc gene and the loxp sequence located downstream of the Terc gene; inserting a Trf1-Dendra2 sequence (SEQ ID No.:3) on the side of the loxp sequence located downstream of the Terc gene facing away from the terminator sequence; through the bone cell specificity Cre conditional knockout of the Terc gene and the terminator sequence which are positioned between the loxp sequences, the Terc gene in bone cells in a mouse can be knocked out, so that the bone cells of the mouse can not synthesize Terc, active telomerase can not be generated in the bone cells, and a mouse model of accelerated aging of the bone cells is obtained; and following the deletion of the Terc gene, the inserted Trf1-Dendra2 sequence was expressed to synthesize Trf1-Dendra2 fluorescent protein to indicate that the Terc gene was deleted.
Description
Technical Field
The invention relates to a technology in the field of genetic engineering, in particular to a method for constructing a bone cell Terc gene knockout mouse model.
Background
Telomeres (Telomere) are DNA-protein complexes present at the ends of chromosomes in eukaryotic cells and function to protect the genome. With the continuous proliferation of cells, telomeres are continuously shortened, and when the protection of the telomeres at the end of chromosomes is lost, an apoptosis mechanism is activated.
Telomerase (Telomerase) is a ribonucleoprotein polymerase with reverse transcription activity in eukaryotic cells, and mainly comprises Telomerase template RNA (Terc), Telomerase reverse transcriptase and Telomerase related proteins. Terc is widely expressed in almost all cells, whereas the expression of telomerase reverse transcriptase is highly regulated, absent or present only at low levels in somatic cells. Telomerase has reverse transcriptase activity, and can synthesize and prolong telomeres by using RNA (telomerase RNA component, Terc) of the telomerase as a template, repair telomere structures and delay cell aging. In most immortalized cell lines, telomerase activity is very high, but studies have shown that increased telomerase activity does not result in cell carcinogenesis, the karyotype and phenotype remain normal, and the number of cell divisions increases dramatically. Telomerase also has effects of resisting apoptosis, regulating cell survival and resisting oxidative stress, protecting mitochondria, and changing energy state of cell; in the field of bone degenerative disease studies, studies have reported a phenotype of src loss, loss of vertebral body bone mass in mice, and indicate that the mechanism of bone mass loss is mainly the inhibition of the bone formation process dominated by aging osteoblasts.
Telomeres exist at the terminal of linear chromosomes in eukaryotic cells in a special structure, mainly consist of G-rich short tandem repeats and binding proteins thereof, mainly expressed as telomere DNA and telomere binding proteins, and the telomere binding proteins mainly comprise telomere repeat binding factor 1 (telomeic repeat binding factor 1, Trf1) and telomere repeat binding factor 2 (telomeic repeat binding factor 2, Trf 2).
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a construction method of a bone cell Terc gene knockout mouse model, which can knock out the bone cell Terc gene in a mouse, so that the bone cell of the mouse can not synthesize Terc, active telomerase can not be generated in the bone cell, and the mouse model with accelerated aging of the bone cell can be obtained; and following the deletion of the Terc gene, the inserted Trf1-Dendra2 sequence was expressed to synthesize Trf1-Dendra2 fluorescent protein to indicate that the Terc gene was deleted.
According to one aspect of the invention, the construction method of the bone cell Terc gene knockout mouse model is provided, and is characterized by comprising the following steps:
performing flox modification on the Terc gene, and respectively inserting a loxp sequence (SEQ ID NO.:1) at the upstream and the downstream of the Terc gene;
inserting a terminator sequence (SEQ ID No.:2) between the Terc gene and the loxp sequence located downstream of the Terc gene;
inserting a Trf1-Dendra2 sequence (SEQ ID No.:3) on the side of the loxp sequence located downstream of the Terc gene facing away from the terminator sequence;
conditional knock-out of the Terc gene and the terminator sequence located between the loxp sequences by osteocyte-specific Cre.
The beneficial effects of the above technical scheme are:
according to the construction method of the bone cell Terc gene knockout mouse model, the Terc gene in bone cells in a mouse can be knocked out, so that the bone cells of the mouse cannot synthesize Terc, active telomerase cannot be generated in the bone cells, and the bone cell accelerated aging mouse model is obtained; and following the knockout of the Terc gene, the inserted Trf1-Dendra2 sequence was expressed to synthesize Trf1-Dendra2 protein to indicate that the Terc gene was knocked out.
And the Trf1-Dendra2 protein expressed and synthesized by the Trf1-Dendra2 sequence can not only be excited to emit green fluorescence under the ordinary state, but also irreversibly emit red fluorescence under the irradiation of ultraviolet light, and the Trf1-Dendra2 protein can be combined into a telomere, so that the telomere can emit green fluorescence, and the length of the telomere can be indicated through the green fluorescence.
After the telomere is irradiated by ultraviolet light, the original telomere shows red fluorescence, and whether the telomere is prolonged or not can be judged.
Further features and advantages of the invention, as well as the structure and operation of various embodiments of the invention, are described in detail below with reference to the accompanying drawings. It should be noted that the invention is not limited to the specific embodiments described herein. These examples are given herein for illustrative purposes only.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, with reference to the accompanying drawings.
FIG. 1 is a schematic diagram of a gene structure.
FIG. 2 is a schematic diagram showing the structure of bone cell gene after the bone cell Terc gene is knocked out.
The features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. Throughout the drawings, like reference numerals designate corresponding elements. In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
As used in this application, the terms "first," "second," and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", and the like are used merely to indicate relative positional relationships, and when the absolute position of the object being described is changed, the relative positional relationships may also be changed accordingly.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.
According to one aspect of the invention, a method for constructing a bone cell Terc gene knockout mouse model is provided.
FIG. 1 is a schematic diagram of the structure of a gene of the present invention. Referring to FIG. 1, the method for constructing a mouse model of bone cell Terc knock-out according to the present invention comprises the following steps A-D. Wherein ncbi of Terc gene is: NR _ 001566.1.
Step A, performing flox modification on the Terc gene, and respectively inserting a loxp sequence (SEQ ID NO: 1) into the upstream and downstream of the Terc gene. Two loxp sequences (SEQ ID No.:1) were inserted upstream and downstream of the Terc gene by existing gene editing techniques, such as CRISPR/Cas9 technique. The presence of loxp sequence should not affect the function of the gene (equivalent to an intron). The loxp sequence is: ataacttcgt atagcataca ttatacgaag ttat are provided.
Step B.inserting a terminator sequence (SEQ ID NO: 2) between the Terc gene and the loxp sequence located downstream of the Terc gene. Terminator sequences (SEQ ID No.:2) were inserted between loxp sequences downstream of the Terc gene by existing gene editing techniques, such as CRISPR/Cas9 technique. A terminator sequence (SEQ ID NO: 2) gcgatgaata aatgaaagct tgcagatctg cgactctaga ggatctgcga ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc cgctacttat ttactttcga acgtctagac gctgagatct cctagacgct gagatctcct agtattagtc ggtatggtgt aaacatctcc aaaatgaacg tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataaaaattttttg gagggtgtgg agggggactt ggactttgta ttttacttac gttaacaaca acaattgaac aaataacgtc gaatattacc aatgtttatt agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatct tcgttatcgt agtgtttaaa gtgtttattt cgtaaaaaaa gtgacgtaag atcaacacca aacaggtttg agtagttaca tagaatagta cagacctagagcgactctag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg cgctgagatc tcctagtatt agtcggtatg gtgtaaacat ctccaaaatg aacgaaattt tttggagggt gtggaggggg acttggactt tgtattttac aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgcttacgttaac aacaacaatt gaacaaataa cgtcgaatat taccaatgtt tatttcgttatcgtagtgtt taaagtgttt atttcgtaaa aaaagtgacg attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctgcgact ctagaggatc ataatcagcc ataccacatt tgtagaggtt taagatcaac accaaacagg tttgagtagt tacatagaat agtacagacc tagacgctga gatctcctag tattagtcgg tatggtgtaa acatctccaa ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tatataggttm aatgaacgaa attttttgga gggtgtggag ggggacttgg actttgtatt ttacttacgt taacaacaac aattgaacaa ataacgtcga atattaccaa acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt tgtttatttc gttatcgtag tgtttaaagt gtttatttcg taaaaaaagt gacgtaagat caacaccaaa caggtttgag tagttacata gaatagtaca ctggatcccc atcaagctga tccggaacct gacctagggg tagttcgact aggccttggt cg is added.
Step C.A Trf1-Dendra2 sequence (SEQ ID No.:3) was inserted on the side of the loxp sequence located downstream of the Terc gene, which faces away from the terminator sequence. The Trf1-Dendra2 sequence is inserted between loxp sequences downstream of the Terc gene by existing gene editing techniques, such as CRISPR/Cas9 techniques. Trf1-Dendra2 sequence (SEQ ID NO: 3) atgtccgtcc tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca gtgccgcgcg ccaagatcca ttcgttgggg gatccgaaca ccccgggaat taacctgatc aaggaggaca tgcgcgtgaa ggtgcacatg gagggcaacg tgaacggcca cgccttcgtg atcgagggcg agggcaaggg caagccctac gagggcaccc agaccgccaa cctgaccgtg aaggagggcg cccccctgcc cttcagctac gacatcctga ccaccgccgt gcactacggc aaccgggtgt tcaccaagta ccccgaggac atccccgact acttcaagca gagcttcccc gagggctaca gctgggagcg caccatgacc ttcgaggaca agggcatctg caccatccgc agcgacatca gcctggaggg cgactgcttc ttccagaacg tgcgcttcaa gggcaccaac ttccccccca acggccccgt gatgcagaag aagaccctga agtgggagcc cagcaccgag aagctgcacg tgcgcgacgg cctgctggtg ggcaacatca acatggccct gctgctggag ggcggcggcc actacctgtg cgacttcaag accacctaca aggccaagaa ggtggtgcag ctgcccgacg cccacttcgt ggaccaccgc atcgagatcc tgggcaacga cagcgactac aacaaggtga agctgtacga gcacgccgtg gcccgctaca gccccctgcc cagccaggtg tgg.
A flox transgenic mouse with the gene structure shown in FIG. 1 was obtained by the steps A-C.
FIG. 2 is a schematic diagram showing the structure of bone cell gene after the bone cell Terc gene is knocked out. Step d. conditional knock-out of the Terc gene and terminator sequences located between loxp sequences by osteocyte specific Cre. The Terc gene and terminator sequences located between the loxp sequences can be knocked out by bone cell specific Cre (dent matrix protein 1, Dmp1 Cre). Specifically, a bone cell Terc gene knockout mouse model having a gene structure shown in fig. 2 can be obtained by breeding a FLOX transgenic mouse and a bone cell specific Cre tool mouse.
Referring again to fig. 1 and 2, between the loxp sequences 101, 104 there is a Terc gene 102 and a terminator sequence 103. The matching of the terminator sequence 103 and the Trf1-Dendra2 sequence 105 can be used for indicating whether the Terc gene 102 in an osteocyte Terc gene knockout mouse model is completely knocked out. After the Terc gene 102 is knocked out, a Trf1-Dendra2 sequence 105 is expressed, and Trf1-Dendra2 fluorescent protein is generated in bone cells, wherein the Trf1-Dendra2 fluorescent protein can emit green fluorescence and can emit red fluorescence under the condition of 405 nm laser. If the Terc gene 102 is not knocked out, the Trf1-Dendra2 sequence 105 cannot be expressed due to the existence of the terminator sequence 103. The Trf1-Dendra2 fluorescent protein synthesized by the Trf1-Dendra2 sequence 105 can emit green fluorescence and can irreversibly emit red fluorescence under the condition of 405-nanometer laser; can also participate in telomere synthesis, namely, participate in telomere synthesis as a substitute of the original telomere repetitive sequence binding factor 1(Trf 1). Thus, under the irradiation of an excitation light source, the Trf1-Dendra2 fluorescent protein can enable the whole telomere to emit green fluorescence intensively, and the length of the telomere can be indicated. Then, after the telomere was irradiated with 405 nm laser light, Trf1-Dendra2 fluorescent protein irreversibly emitted red fluorescence. After the telomere is irradiated by 405 nm laser, if the telomere is further extended, the extended part emits green fluorescence, and the green fluorescence can be used for indicating whether the telomere is extended.
In conclusion, the construction method of the bone cell Terc gene knockout mouse model can knock out the Terc gene in bone cells of a mouse, so that the bone cells of the mouse cannot synthesize Terc, telomerase cannot be generated in the bone cells, and the bone cell accelerated aging mouse model is obtained; and following the knockout of the Terc gene, the inserted Trf1-Dendra2 sequence was expressed to synthesize Trf1-Dendra2 protein to indicate that the Terc gene was knocked out.
And besides being capable of exciting to emit green fluorescence under a common state, the Trf1-Dendra2 protein expressed and synthesized by the Trf1-Dendra2 sequence can irreversibly emit red fluorescence under the irradiation of 405-nanometer laser, and the Trf1-Dendra2 protein can be combined into a telomere, so that the telomere can emit green fluorescence, and the length of the telomere can be indicated through the green fluorescence.
After the telomere is irradiated by 405 nm laser, the original telomere shows red fluorescence, and whether the telomere is prolonged can be further judged.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
SEQUENCE LISTING
<110> national institute of Ningbo Life and health industry
<120> construction method of bone cell Terc gene knockout mouse model
<130>
<160> 3
<170> PatentIn version 3.5
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<211> 34
<212> DNA
<213> Artificial Sequence
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<223> loxp sequence
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ataacttcgt atagcataca ttatacgaag ttat 34
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<211> 1663
<212> DNA
<213> Artificial Sequence
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<223> terminator sequence
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gcgatgaata aatgaaagct tgcagatctg cgactctaga ggatctgcga ctctagagga 60
tcataatcag ccataccaca tttgtagagg ttttacttgc cgctacttat ttactttcga 120
acgtctagac gctgagatct cctagacgct gagatctcct agtattagtc ggtatggtgt 180
aaacatctcc aaaatgaacg tttaaaaaac ctcccacacc tccccctgaa cctgaaacat 240
aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa 300
aaattttttg gagggtgtgg agggggactt ggactttgta ttttacttac gttaacaaca 360
acaattgaac aaataacgtc gaatattacc aatgtttatt agcaatagca tcacaaattt 420
cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 480
atcttatcat gtctggatct tcgttatcgt agtgtttaaa gtgtttattt cgtaaaaaaa 540
gtgacgtaag atcaacacca aacaggtttg agtagttaca tagaatagta cagacctaga 600
gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa 660
aaacctccca cacctccccc tgaacctgaa acataaaatg cgctgagatc tcctagtatt 720
agtcggtatg gtgtaaacat ctccaaaatg aacgaaattt tttggagggt gtggaggggg 780
acttggactt tgtattttac aatgcaattg ttgttgttaa cttgtttatt gcagcttata 840
atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 900
ttacgttaac aacaacaatt gaacaaataa cgtcgaatat taccaatgtt tatttcgtta 960
tcgtagtgtt taaagtgttt atttcgtaaa aaaagtgacg attctagttg tggtttgtcc 1020
aaactcatca atgtatctta tcatgtctgg atctgcgact ctagaggatc ataatcagcc 1080
ataccacatt tgtagaggtt taagatcaac accaaacagg tttgagtagt tacatagaat 1140
agtacagacc tagacgctga gatctcctag tattagtcgg tatggtgtaa acatctccaa 1200
ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca 1260
attgttgttg ttaacttgtt tattgcagct tataatggtt maatgaacga aattttttgg 1320
agggtgtgga gggggacttg gactttgtat tttacttacg ttaacaacaa caattgaaca 1380
aataacgtcg aatattacca aacaaataaa gcaatagcat cacaaatttc acaaataaag 1440
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 1500
ttgtttattt cgttatcgta gtgtttaaag tgtttatttc gtaaaaaaag tgacgtaaga 1560
tcaacaccaa acaggtttga gtagttacat agaatagtac actggatccc catcaagctg 1620
atccggaacc tgacctaggg gtagttcgac taggccttgg tcg 1663
<210> 3
<211> 783
<212> DNA
<213> Artificial Sequence
<220>
<223> Trf1-Dendra2 sequence
<400> 3
atgtccgtcc tgacgccgct gctgctgcgg ggcttgacag gctcggcccg gcggctccca 60
gtgccgcgcg ccaagatcca ttcgttgggg gatccgaaca ccccgggaat taacctgatc 120
aaggaggaca tgcgcgtgaa ggtgcacatg gagggcaacg tgaacggcca cgccttcgtg 180
atcgagggcg agggcaaggg caagccctac gagggcaccc agaccgccaa cctgaccgtg 240
aaggagggcg cccccctgcc cttcagctac gacatcctga ccaccgccgt gcactacggc 300
aaccgggtgt tcaccaagta ccccgaggac atccccgact acttcaagca gagcttcccc 360
gagggctaca gctgggagcg caccatgacc ttcgaggaca agggcatctg caccatccgc 420
agcgacatca gcctggaggg cgactgcttc ttccagaacg tgcgcttcaa gggcaccaac 480
ttccccccca acggccccgt gatgcagaag aagaccctga agtgggagcc cagcaccgag 540
aagctgcacg tgcgcgacgg cctgctggtg ggcaacatca acatggccct gctgctggag 600
ggcggcggcc actacctgtg cgacttcaag accacctaca aggccaagaa ggtggtgcag 660
ctgcccgacg cccacttcgt ggaccaccgc atcgagatcc tgggcaacga cagcgactac 720
aacaaggtga agctgtacga gcacgccgtg gcccgctaca gccccctgcc cagccaggtg 780
tgg 783
Claims (1)
1. A method for constructing a bone cell Terc gene knockout mouse model is characterized by comprising the following steps:
performing flox modification on the Terc gene, and respectively inserting a loxp sequence (SEQ ID NO.:1) at the upstream and the downstream of the Terc gene;
inserting a terminator sequence (SEQ ID No.:2) between the Terc gene and the loxp sequence located downstream of the Terc gene;
inserting a Trf1-Dendra2 sequence (SEQ ID No.:3) on the side of the loxp sequence located downstream of the Terc gene facing away from the terminator sequence;
conditional knock-out of the Terc gene and the terminator sequence located between the loxp sequences by osteocyte-specific Cre.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277613B1 (en) * | 1998-06-10 | 2001-08-21 | The Rockefeller University | TRF1 binding protein, methods of use thereof |
| US20140024066A1 (en) * | 2012-06-20 | 2014-01-23 | California Institute Of Technology | Animal model having photo-activatable mitochondria |
| KR20160032359A (en) * | 2014-09-15 | 2016-03-24 | 서강대학교산학협력단 | A Method for Inhibition of Pluripotent stem cell-derived Teratoma Formation |
| CN107674879A (en) * | 2016-08-01 | 2018-02-09 | 深圳先进技术研究院 | A kind of photogene plasmid and its application |
| US20180064746A1 (en) * | 2015-03-19 | 2018-03-08 | The Johns Hopkins University | Assay for telomere length regulators |
| US20180230444A1 (en) * | 2015-08-10 | 2018-08-16 | Cellivery Therapeutics, Inc. | Cell-permeable cre (icp-cre) recombinant protein and use thereof |
| CN109844124A (en) * | 2016-05-20 | 2019-06-04 | 哈佛学院董事及会员团体 | The gene therapy method of age-related disease and illness |
| CN112824529A (en) * | 2019-11-21 | 2021-05-21 | 北京大学 | Conditional gene knockout or rescue method |
| CN113293140A (en) * | 2021-05-25 | 2021-08-24 | 昆明理工大学 | Telomerase negative mouse ALT cell model and construction method thereof |
| CN113678789A (en) * | 2021-08-26 | 2021-11-23 | 嘉兴学院 | Mir-379/410 gene cluster knockout mouse model and construction method thereof |
| CN115120194A (en) * | 2022-06-09 | 2022-09-30 | 国科宁波生命与健康产业研究院 | Fluorescent protein labeled mouse monitoring method, system, device and medium |
-
2022
- 2022-06-09 CN CN202210654204.1A patent/CN114921480A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277613B1 (en) * | 1998-06-10 | 2001-08-21 | The Rockefeller University | TRF1 binding protein, methods of use thereof |
| US20140024066A1 (en) * | 2012-06-20 | 2014-01-23 | California Institute Of Technology | Animal model having photo-activatable mitochondria |
| KR20160032359A (en) * | 2014-09-15 | 2016-03-24 | 서강대학교산학협력단 | A Method for Inhibition of Pluripotent stem cell-derived Teratoma Formation |
| US20180064746A1 (en) * | 2015-03-19 | 2018-03-08 | The Johns Hopkins University | Assay for telomere length regulators |
| US20180230444A1 (en) * | 2015-08-10 | 2018-08-16 | Cellivery Therapeutics, Inc. | Cell-permeable cre (icp-cre) recombinant protein and use thereof |
| CN109844124A (en) * | 2016-05-20 | 2019-06-04 | 哈佛学院董事及会员团体 | The gene therapy method of age-related disease and illness |
| CN107674879A (en) * | 2016-08-01 | 2018-02-09 | 深圳先进技术研究院 | A kind of photogene plasmid and its application |
| CN112824529A (en) * | 2019-11-21 | 2021-05-21 | 北京大学 | Conditional gene knockout or rescue method |
| CN113293140A (en) * | 2021-05-25 | 2021-08-24 | 昆明理工大学 | Telomerase negative mouse ALT cell model and construction method thereof |
| CN113678789A (en) * | 2021-08-26 | 2021-11-23 | 嘉兴学院 | Mir-379/410 gene cluster knockout mouse model and construction method thereof |
| CN115120194A (en) * | 2022-06-09 | 2022-09-30 | 国科宁波生命与健康产业研究院 | Fluorescent protein labeled mouse monitoring method, system, device and medium |
Non-Patent Citations (4)
| Title |
|---|
| HAMID SAEED 等: "Telomerase-deficient mice exhibit bone loss owing to defects in osteoblasts and increased osteoclastogenesis by inflammatory microenvironment", JOURNAL OF BONE AND MINERAL RESEARCH HOMEPAGE, vol. 26, no. 7, pages 1495 * |
| MARIA A.BLASCO 等: "Immunosenescence phenotypes in the telomerase knockout mouse", SPRING SEMINARS IN IMMUNOPATHOLOGY, vol. 24, pages 75 - 85 * |
| 冷潇等: "Lck-Cre×Perpflox/flox条件敲除小鼠的繁育及基因型鉴定", 成都医学院学报, vol. 9, no. 2, pages 1 - 2 * |
| 王强 等: "干细胞移植治疗骨质疏松", 中华骨质疏松和骨矿盐疾病杂志, vol. 5, no. 3, pages 225 - 229 * |
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