CN110438086A - NDUFS3 Knockdown and/or the steady of overexpression turn strain and its construction method - Google Patents
NDUFS3 Knockdown and/or the steady of overexpression turn strain and its construction method Download PDFInfo
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- 101001128687 Homo sapiens NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial Proteins 0.000 title claims abstract description 54
- 102000050479 human NDUFS3 Human genes 0.000 title claims abstract description 54
- 238000010276 construction Methods 0.000 title claims abstract description 52
- 230000002018 overexpression Effects 0.000 title claims abstract description 34
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims abstract description 48
- 238000001890 transfection Methods 0.000 claims abstract description 44
- 241000700605 Viruses Species 0.000 claims abstract description 37
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- 238000003197 gene knockdown Methods 0.000 claims abstract 17
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Abstract
The invention discloses NDUFS3 Knockdowns and/or the steady of overexpression to turn strain and its construction method, it is related to gene engineering technology field, specifically, the construction method includes that target gene is inserted into cell strain, the cell strain after transfection is screened by using 8~10 μ g/mL puromycins, NDUFS3 Knockdown is obtained and/or the steady of overexpression turns strain.Relative to traditional stable cell strain construction method, construction method provided in an embodiment of the present invention reduces and optimizes experimental procedure, improve transfection efficiency, the virus of disposable selection high titre is transfected, the drug screening of high concentration is carried out later, reduce experimental period, screening time can be foreshortened to 5 days, and the puromycin for not needing low concentration is maintained.
Description
Technical field
The present invention relates to gene engineering technology field, in particular to the steady of NDUFS3 Knockdown and/or overexpression
Turn strain and its construction method.
Background technique
Traditional stable cell strain construction method, it usually needs preliminary experiment detect each plant of cell MOI value and purine it is mould
Then plain half lethal concentration calculates virus concentration when cell transfecting according to MOI value, dense according to the median lethal of puromycin
Degree carries out drug screening, and the MOI value and puromycin half lethal concentration of each plant of cell are different from, so that every transfection one
The new cell of strain, requires to carry out cumbersome preliminary experiment.
Traditional stable cell strain construction method after virus transfection 72 hours, usually begins to use preliminary experiment to obtain fast
Purine mycin half lethal concentration carries out drug screening, and replacement has the culture medium of puromycin daily, at least screening 14 days, later
The puromycin of low concentration is needed to carry out maintaining at least one moon, also to guarantee the transfection efficiency of stable cell strain.
In addition, traditional stable cell strain construction method, is usually only capable of being up to the transfection efficiency of stable cell strain
90%, unless screening such complicated and unworkable experimental method using limiting dilution and monoclonal, it can be only achieved 100%
Transfection efficiency.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide the construction method for surely turning strain of NDUFS3 Knockdown and/or overexpression,
The stable cell strain of NDUFS3 Knockdown and the stable cell strain of NDUFS3 gene overexpression, construction method behaviour of the invention
Work is simple, the used time is short, transfection efficiency is high.
The present invention is implemented as follows:
In a first aspect, the construction method for surely turning strain of a kind of NDUFS3 Knockdown and/or overexpression comprising: by mesh
Gene into cells strain, the cell strain after transfection is screened at least once using 8~10 μ g/mL puromycins, is obtained
NDUFS3 Knockdown and/or the steady of overexpression turn strain.
Preferably, four screenings are carried out to the cell strain after transfection using 8~10 μ g/mL puromycins；
Preferably, the interval time between adjacent screening is 10~14 hours；
Preferably, screen to cell strain luciferase expression rate >=90%, preferably >=95%, more preferably 100%, obtain
NDUFS3 Knockdown and/or the steady of overexpression turn strain；
Preferably, target gene is imported into (1~9) × 103The cell strain suspension of a/mL.
The embodiment of the present invention does not need to detect its MOI value and puromycin half for different cell strain progress preliminary experiments
Lethasl concentration, the disposable virus for selecting high titre are transfected, and carry out the drug screening of high concentration later.
On the other hand, construction method provided in an embodiment of the present invention reduces experimental period, uses high concentration puromycin
Pressure screening, can foreshorten to screening time 5 days, and the puromycin for not needing low concentration is maintained.
Further more, relatively traditional stable cell strain construction method, construction method provided in an embodiment of the present invention improve surely
The transfection efficiency for turning cell strain finally may be used by the drug screening of the virus transfection and 4 high concentration puromycins of a high concentration
So that stable cell strain transfection efficiency reaches 100%.
Specifically, above-mentioned NDUFS3 Knockdown and/or overexpression surely to turn the construction method of strain include NDUFS3 gene
The construction method for surely turning strain of the construction method for surely turning strain and/or NDUFS3 Knockdown that are overexpressed.
It should be noted that and/or indicating the construction method packet for surely turning strain of NDUFS3 Knockdown and/or overexpression
It includes: the construction method for surely turning strain of NDUFS3 gene overexpression or the construction method for surely turning strain of NDUFS3 Knockdown, or it is same
When include this strike it is low and be overexpressed two kinds of construction methods.
Specifically, the construction method for surely turning strain of above-mentioned NDUFS3 gene overexpression includes: using such as SEQ ID No.1 institute
The upstream primer and the downstream primer as shown in SEQ ID No.2 shown expand target gene, by the mesh after amplification
Gene into cells strain, screened at least once by puromycin, obtain be overexpressed NDUFS3 stable cell strain.
Preferably, the target gene after amplification is imported cell strain includes: that target gene is inserted into slow virus carrier,
Then transfection cell strain.
The construction method for surely turning strain of above-mentioned NDUFS3 Knockdown includes: corresponding according to low target sequence synthesis is struck
ShRNA, packs corresponding slow virus, after transfection cell strain, is screened at least once by puromycin, obtains striking low
NDUFS3 gene stable cell strain；
Strike low target sequence include the first target spot shown in SEQ ID No.3, the second target spot shown in SEQ ID No.4 and
One or more of third target spot shown in SEQ ID No.5.
It is in alternative embodiments, described that strike low target sequence be preferably the first target spot shown in SEQ ID No.3.Root
Effect, significant effect are turned down with mRNA level in-site and protein level according to the low NDUFS3 gene that strikes that the first target spot carries out.
It in alternative embodiments, include: that basis strikes low target spot sequence according to striking low target sequence to synthesize corresponding shRNA
Column design primer pair makes annealing treatment the primer pair, and single-stranded DNA is made to be annealed into shRNA；
The upstream primer sequence of first target spot is as shown in SEQ ID No.6, the downstream primer sequence of first target spot
Column are as shown in SEQ ID No.7, and the upstream primer sequence of second target spot is as shown in SEQ ID No.8, second target spot
Downstream primer sequence as shown in SEQ ID No.9, the upstream primer sequence of the third target spot as shown in SEQ ID No.10,
The downstream primer sequence of the third target spot is as shown in SEQ ID No.11；
Preferably, the annealing includes then cooling to room temperature 85~95 DEG C of 10~20min of heating of the primer pair.
In alternative embodiments, packing corresponding slow virus includes: that the product after the annealing is inserted into slow virus
In carrier, competent cell is transfected, virus liquid is obtained；
Preferably, the slow virus carrier includes GV493 or GV492；GV493 or GV492 please refers to attached drawing 1.
Preferably, the competent cell is 293T cell；
Preferably, the inoculum density of the competent cell is (1~9) × 103A/mL.
Transfection cell strain includes: by the virus liquid transfection cell strain, and when transfection, virus titer is (1~9) E+8TU/
mL。
Transfection carries out in the presence of transfecting reinforcing agent；
Preferably, the transfection reinforcing agent includes Polybrene；
Preferably, the Polybrene concentration is 4~8 μ g/mL.
Cell strain includes A875 cell, A375 cell and/or SK-MEL-110 cell.
Second aspect, present invention implementation additionally provides a kind of stable cell strain of NDUFS3 Knockdown, by above-mentioned
NDUFS3 Knockdown and/or the construction method for surely turning strain of overexpression obtain.
The third aspect, present invention implementation additionally provides a kind of stable cell strain of NDUFS3 gene overexpression, by above-mentioned
NDUFS3 Knockdown and/or overexpression surely turn the construction method of strain and obtain.
The invention has the following advantages:
The embodiment of the invention provides the construction method for surely turning strain of a kind of NDUFS3 Knockdown and/or overexpression,
Include: that target gene is inserted into cell strain, the cell strain after transfection screened by using 8~10 μ g/mL puromycins,
It obtains NDUFS3 Knockdown and/or the steady of overexpression turns strain.Relative to traditional stable cell strain construction method, the present invention is real
The construction method for applying example offer reduces and optimizes experimental procedure, improves transfection efficiency, the disposable virus for selecting high titre
It is transfected, carries out the drug screening of high concentration later.Reduce experimental period, screening time can be foreshortened to 5 days, and be not required to
The puromycin of low concentration is wanted to be maintained.
In addition, the embodiment of the invention also provides the NDUFS3 Knockdown constructed by above-mentioned construction method and/or
What is be overexpressed surely turns strain, this surely turns the overexpression or strike low NDUFS3 gene that strain can be stable.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the plasmid map in summary of the invention；Wherein, the plasmid map that A is GV492 in Fig. 1, B is GV493 in Fig. 1
Plasmid map；
Fig. 2 is that building A875, SK-MEL-110 cell NDUFS3 is overexpressed and strikes low stable cell strain sequencing in embodiment 1
Result figure；
Fig. 3 is the phase contrast microscope and fluorescence microscope result of the stable cell strain in embodiment 1；Wherein, in Fig. 3
A is each stable cell strain phase contrast microscope of A875 and fluorescence microscope picture in embodiment 1, and B is SK-MEL-110 in Fig. 3
Each stable cell strain phase contrast microscope and fluorescence microscope picture；
Fig. 4 is the qualification result that NDUFS3 was overexpressed or struck low melanoma stable cell strain in embodiment 1；
Fig. 5 is that conventional method and optimization method construct the time that stable cell strain needs in comparative example 1；
Fig. 6 surely turns NDUFS3 gene overexpression cell strain micro- for conventional method in comparative example 1 and optimization method building
Transfection efficiency under mirror；
Fig. 7 is that conventional method and optimization method building surely turn NDUFS3 Knockdown cell strain in microscope in comparative example 1
Under transfection efficiency.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
Surely turn the steady of strain and NDUFS3 Knockdown or overexpression the present invention provides a kind of NDUFS3 gene overexpression
Turn the construction method of strain comprising following steps:
Kind plate
Disappear in logarithmic growth phase human melanoma cell strain, including cell strain SK-MEL-110, A875, A375, pancreatin
Change, is made 5 × 10 with complete medium3A/mL cell suspension, is seeded in 6 orifice plates.It waits cell adherent and starts division and increase
It grows.
Infectious titer transfection
Target gene is imported into cell strain:
A) be overexpressed slow virus package carrier: the primer sequence for expressing target gene includes as shown in SEQ ID No.1
Upstream primer and the downstream primer as shown in SEQ ID No.2.Using the upstream primer as shown in SEQ ID No.1 and such as SEQ
Downstream primer shown in ID No.2 carries out PCR amplification to target gene.The sequence of target gene such as UniProtKB-O75489
(NDUS3_HUMAN) cDNA sequence shown in (transcript of the cDNA sequence with reference to NM_004551, transcript NM_
004551.3 corresponding peptide chain is NP_004542.1, and NP_004542.1 corresponding is isoform1) shown in.By the mesh after amplification
Gene connect and (by BamHI/AgeI digestion, then connect under the action of T4 DNA ligase with slow virus carrier GV492
It connects.), building plasmid is overexpressed slow virus package carrier GV492 (A in Fig. 1), obtains GV492-NDUFS3 (25396-1) carrier.
B) strike low slow virus package carrier: striking low target sequence includes the first target spot, such as SEQ shown in SEQ ID No.3
Second target spot shown in ID No.4 and the third target spot as shown in SEQ ID No.5.According to striking low target sequence design primer
It is right, the primer pair is made annealing treatment, single-stranded DNA is made to be annealed into shRNA.Annealing includes adding 90 DEG C of the primer pair
Hot 15min, then cools to room temperature.
The upstream primer sequence of first target spot is as shown in SEQ ID No.6, and the downstream primer sequence of first target spot is such as
Shown in SEQ ID No.7, the upstream primer sequence of second target spot is as shown in SEQ ID No.8, under second target spot
Primer sequence is swum as shown in SEQ ID No.9, the upstream primer sequence of the third target spot is described as shown in SEQ ID No.10
The downstream primer sequence of third target spot is as shown in SEQ ID No.11.
By in the product insertion slow virus carrier GV493 after the annealing, obtains plasmid and strike low slow virus package carrier
GV493 (B in Fig. 1), specially GV493-NDUFS3-RNAi (59731-11), GV493-NDUFS3-RNAi (59732-1) and
GV493-NDUFS3-RNAi(59733-11)。
With above-mentioned overexpression slow virus package carrier and strike low slow virus package carrier and pHelper1.0 carrier respectively
PHelper2.0 carrier transfects 293T incasing cells jointly, and transfection is changed to the DMEM complete culture solution containing 10%FBS afterwards for 24 hours
Continue to cultivate 48h, collect all supernatants of culture cell, 4 DEG C, 500rpm is centrifuged 10min, to remove floating cells and its broken
Piece, supernatant obtains slow virus stoste after being filtered with 0.45 μm of PVDF filter device after centrifugation, and slow virus stoste is respectively as follows: LV-
NDUFS3 (25396-1) over-express vector, LV-NDUFS3-RNAi (59731-1) shRNA carrier, LV-NDUFS3-RNAi
(59732-1) shRNA carrier, GV493 mediate LV-NDUFS3-RNAi (59733-1) shRNA carrier.
Obtained slow virus package carrier is sequenced, attached drawing 2 is please referred to, shows vector construction success.
For kind after plate about 48 hours, preparation is added in every empty Opti-MEM culture medium 600 μ L, every hole addition Polybrene of being added
Good infectious titer.Final conversion, makes virus titer need to reach 1E+8TU/mL, Polybrene concentration need to be up to 5 μ g/mL.
High concentration puromycin pressure drug screening
After virus transfection 48 hours, fluorescence microscopy microscopic observation luciferase expression situation please refers to the (A and Fig. 3 in Fig. 3 of attached drawing 3
Middle B), when the estimation of luciferase expression rate reaches 70%, carrying out replacement virus liquid is Opti-MEM culture medium, and Opti- is added in every hole
MEM culture medium 2mL continued culture to 72 hours.
Puromycin is added in every hole later, and puromycin concentration is made to reach 10 μ g/mL.Every 12 hours later replacement Opti-
MEM culture medium 2mL, while puromycin is added, and puromycin concentration is made to reach 10 μ g/mL.Continue every 12 hours so more
The Opti-MEM culture medium added with the puromycin of high concentration is changed, is amounted to 4 times.
To the 5th day (120 hours) of transfection, under the microscope, the luciferase expression rate of cell strain can reach 100% to fluorescence microscopy,
Prompt stable cell strain transfection efficiency reaches 100%.
Carry out q-PCR and Western-blot identification:
Above-mentioned cell strain RNA and protein are collected, q-PCR and Western-blot is carried out to NDUFS3 gene and is identified, inspection
It surveys result and please refers to attached drawing 4.
In Humanmachine tumour A875 cell, compared with the control group, after being overexpressed NDUFS3, in NDUFS3 mRNA level in-site
230% (p < 0.0001) is risen, protein level rises 78.8% (p=0.0019)；After striking low NDUFS3, NDUFS3 mRNA
Level has dropped 60.4% (p < 0.0001), and protein level has dropped 78.7% (p=0.0105) (A1~A3 in Fig. 4).In people
In melanoma SK-MEL-110 cell, compared with the control group, after being overexpressed NDUFS3, NDUFS3mRNA level rises 197
Again (p < 0.0001), protein level rises 165% (p < 0.0001)；After striking low NDUFS3, NDUFS3 mRNA level in-site is had dropped
62.4% (p < 0.0001), protein level have dropped 85.6% (p < 0.0001) (B1~B3 in Fig. 4).The above results suggest that In
In human melanoma cell A875 and SK-MEL-110 cell, NDUFS3, which is overexpressed and strikes, low surely to be turned strain and constructs successfully.
It secondary culture and freezes:
Above-mentioned stable cell strain is subjected to secondary culture and is frozen.
Comparative example 1
Verify the effect for the construction method that the embodiment of the present invention 1 provides.
The construction method provided using embodiment 1 is to tri- kinds of human melanoma cell strains of A875, A375, SK-mel-110
It NDUFS3 gene overexpression and strikes low stable cell strain and is constructed, comparison one group of reference examples of setting, reference examples and embodiment 1
Difference is that the screening of puromycin is different, distinguishes as follows:
After virus transfection 72 hours, the puromycin half lethal concentration for usually beginning to use preliminary experiment to obtain carries out drug
Screening, replacement has the culture medium of puromycin daily, at least screens 14 days, the puromycin of low concentration is also needed to carry out later
At least one moon is maintained, to guarantee the transfection efficiency of stable cell strain.
As shown in Fig. 5, transfection efficiency the time required to calculating the construction method that embodiment 1 provides and the construction method of reference examples
Test result is as shown in Figure 6 and Figure 7.
As shown in Figure 5, embodiment 1 can foreshorten to screening time 5 days, and the puromycin for not needing low concentration carries out
It maintains.Using conventional method (traditional method) and optimization method, (optimization method is implemented for we
Example 1), it compared tri- kinds of human melanoma cell strain NDUFS3 gene overexpressions of A875, A375, SK-mel-110 and strike low steady turn
The time of time required for cell strain constructs, discovery conventional method building stable cell strain are longer than optimization side provided by the present application
Method, difference have statistical significance, and P value is less than 0.01.
By Fig. 6 and 7 it is found that screening technique and traditional screening technique in embodiment 1, compares A875, A375, SK-
Tri- kinds of human melanoma cell strain NDUFS3 gene overexpressions of mel-110 and strike low stable cell strain strike it is low or be overexpressed effect
Rate finds that the transfection efficiency of optimization method is higher than conventional method (the GFP positive cell of optimization method is significantly more than conventional method), tool
There is statistical difference (P value is less than 0.05).
To sum up, the embodiment of the invention provides a kind of NDUFS3 Knockdown and/or the building sides for surely turning strain of overexpression
Method comprising: target gene is inserted into cell strain, the cell strain after transfection is carried out by using 8~10 μ g/mL puromycins
Screening, obtains NDUFS3 Knockdown and/or the steady of overexpression turns strain.Relative to traditional stable cell strain construction method, originally
The construction method that inventive embodiments provide reduces and optimizes experimental procedure, improves transfection efficiency, disposably selects high titre
Virus transfected, later carry out high concentration drug screening.Reduce experimental period, screening time can be foreshortened to 5 days,
And the puromycin for not needing low concentration is maintained.
In addition, the embodiment of the invention also provides the NDUFS3 Knockdown constructed by above-mentioned construction method and/or
What is be overexpressed surely turns strain, this surely turns the overexpression or strike low NDUFS3 gene that strain can be stable.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. the construction method for surely turning strain of a kind of NDUFS3 Knockdown and/or overexpression, characterized in that it comprises: by mesh
Gene into cells strain, the cell strain after transfection is screened at least once using 8~10 μ g/mL puromycins, is obtained
NDUFS3 Knockdown and/or the steady of overexpression turn strain；
Preferably, four screenings are carried out to the cell strain after transfection using 8~10 μ g/mL puromycins；
Preferably, the interval time between adjacent screening is 10~14 hours；
Preferably, screen to cell strain luciferase expression rate >=90%, preferably >=95%, more preferably 100%, obtain NDUFS3
Knockdown and/or the steady of overexpression turn strain；
Preferably, target gene is imported into (1~9) × 103The cell strain suspension of a/mL.
2. NDUFS3 Knockdown and/or the construction method for surely turning strain of overexpression, feature exist according to claim 1
In the screening time for using puromycin to screen is 4~6 day.
3. NDUFS3 Knockdown and/or the construction method for surely turning strain of overexpression according to claim 1, the building side
Method includes the construction method for surely turning strain of NDUFS3 gene overexpression and/or the building side for surely turning strain of NDUFS3 Knockdown
Method；
The construction method for surely turning strain of the NDUFS3 gene overexpression includes: to be drawn using the upstream as shown in SEQ ID No.1
Object and the downstream primer as shown in SEQ ID No.2 expand target gene, and the target gene after amplification is imported
Cell strain screens cell strain by 8~10 μ g/mL puromycins at least once, obtains overexpression NDUFS3 and surely turns thin
Born of the same parents' strain；
The construction method for surely turning strain of the NDUFS3 Knockdown includes: that basis strikes the corresponding shRNA of low target sequence synthesis,
It packs corresponding slow virus, after transfection cell strain, cell strain is screened at least once by 8~10 μ g/mL puromycins,
It obtains striking low NDUFS3 gene stable cell strain；
It is described that strike low target sequence include the first target spot shown in SEQ ID No.3, the second target spot as shown in SEQ ID No.4
One or more of with the third target spot as shown in SEQ ID No.5.
4. the construction method for surely turning strain of NDUFS3 Knockdown according to claim 3 and/or overexpression, feature exist
In it includes: that basis strikes low target sequence design primer pair that the basis, which strikes low target sequence and synthesizes corresponding shRNA, to described
Primer pair is made annealing treatment, and single-stranded DNA is made to be annealed into shRNA；
The upstream primer sequence of first target spot is as shown in SEQ ID No.6, and the downstream primer sequence of first target spot is such as
Shown in SEQ ID No.7, the upstream primer sequence of second target spot is as shown in SEQ ID No.8, under second target spot
Primer sequence is swum as shown in SEQ ID No.9, the upstream primer sequence of the third target spot is described as shown in SEQ ID No.10
The downstream primer sequence of third target spot is as shown in SEQ ID No.11；
Preferably, the annealing includes then cooling to room temperature 85~95 DEG C of 10~20min of heating of the primer pair.
5. the construction method for surely turning strain of NDUFS3 Knockdown according to claim 3 and/or overexpression, feature exist
In the corresponding slow virus of packaging includes: by the product insertion slow virus carrier after the annealing, and transfection competence is thin
Born of the same parents obtain virus liquid；
Preferably, the slow virus carrier includes GV493 or GV492；
Preferably, the competent cell is 293T cell；
Preferably, the inoculum density of the competent cell is (1~9) × 103A/mL.
6. the construction method for surely turning strain of NDUFS3 Knockdown according to claim 5 and/or overexpression, feature exist
It include: by the virus liquid transfection cell strain in, the transfection cell strain, when transfection, virus titer is (1~9) E+8TU/mL.
7. the construction method for surely turning strain of NDUFS3 Knockdown according to claim 6 and/or overexpression, feature exist
In the transfection carries out in the presence of transfecting reinforcing agent；
Preferably, the transfection reinforcing agent includes Polybrene；
Preferably, the Polybrene concentration is 4~8 μ g/mL.
8. the building side for surely turning strain of described in any item NDUFS3 Knockdowns and/or overexpression according to claim 1~7
Method, which is characterized in that the cell strain includes A875 cell, A375 cell and/or SK-MEL-110 cell.
9. a kind of stable cell strain of NDUFS3 Knockdown, which is characterized in that it is by according to any one of claims 1 to 8
NDUFS3 Knockdown and/or the construction method for surely turning strain of overexpression obtain.
10. a kind of stable cell strain of NDUFS3 gene overexpression, which is characterized in that it is as described in any one of claim 1~8
NDUFS3 Knockdown and/or overexpression surely turn the construction method of strain and be prepared.
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| WO2012103455A1 (en) * | 2011-01-28 | 2012-08-02 | Pangea Biosciences, Inc. | Biomarkers and their uses in cancer detection and therapy |
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