CN108676855B - Method and primer for identifying bovine transgenic copy number by standard curve fluorescent quantitative PCR (polymerase chain reaction) - Google Patents
Method and primer for identifying bovine transgenic copy number by standard curve fluorescent quantitative PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention discloses a method and primers for identifying bovine transgenic copy number by standard curve fluorescent quantitative PCR. The detection method is characterized in that a real-time fluorescent quantitative PCR method is utilized, and constructed transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (transgenosis type) and non-transformed bovine 4-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (wild type) are used as detection objects. Respectively detecting transferred bovine four-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (transgenosis type) and non-transferred bovine 4-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (wild type), and directly detecting whether a sample to be detected is transferred into bovine 4-factor Oct4, Sox2, Klf4 and cMyc genes and the transgenic copy number thereof. Lays a foundation for genetic stability screening experiments of transgenic cattle.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method and primers for identifying bovine transgenic copy number by standard curve fluorescent quantitative PCR.
Background
Transgenic animals are animals in which a known exogenous gene is introduced into the genome of an animal and integrated into the chromosome by various experimental methods, and the exogenous gene can be stably transmitted to the offspring. Quantitative detection of copy number of exogenous gene is one of important contents for understanding and evaluating transgenic animals. Through carrying out early detection on the transgenic animal, the basic characteristics of the transgenic animal are known in time, an evaluation basis is provided for later cultivation, development and utilization of the transgenic animal, and waste of manpower, material resources and financial resources is avoided. The traditional method for detecting the copy number of the exogenous gene is Southern blot, but is time-consuming, labor-consuming and requires a large amount of genomic DNA. The real-time fluorescence quantitative PCR technology is a DNA/RNA quantitative technology which is rapidly developed in recent years, has the advantages of high specificity, high signal-to-noise ratio, high safety, high efficiency, high throughput, low cost and the like compared with Southern blot, and is widely applied to the detection of the copy number of a transgenic exogenous gene.
The Wangxiaojian (2007) and other reports show that the whole process can be completed within 2h by using real-time fluorescence quantitative PCR to detect the CARK transgenic mice, the copy numbers of the exogenous genes of 3 mice are respectively 1, 17 and 45, the result is completely consistent with the result of Southern hybridization, the delta Ct of 3 repeated experiments is very stable, and the SD is only 0.15-0.46. The method comprises the steps of detecting the copy number of an external source gene of an antithrombin III transgenic goat by using real-time fluorescent quantitative PCR (polymerase chain reaction) reported by Yanjunshan (2010) and the like, respectively obtaining standard curve equations of ATIII and GAPDH by making standard curves of the external source gene ATIII and an internal reference gene GAPDH, and calculating the copy number of the external source gene of the ATIII transgenic goat. Plum Shaolin (2013) and other reports utilize real-time fluorescence quantitative detection for nestin-GFP transgenic mouse exogenous gene integration copy number, the expression of the GFP gene of the transgenic mouse is consistent with that of the nestin gene, and the GFP gene copy number of the transgenic mouse is detected through real-time fluorescence quantitative PCR, so that the obtained transgenic mouse exogenous gene copy number is respectively 3 and 4. Taverniers (2005) reported that the accuracy of this method was completely matched with the Southern hybridization results by detecting 4 transgenic animals using real-time fluorescent quantitative PCR.
Disclosure of Invention
The invention aims to provide a method and primers for identifying bovine transgenic copy number by standard curve fluorescent quantitative PCR. The detection method is characterized in that a real-time fluorescent quantitative PCR method is utilized, and constructed transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (transgenosis type) and non-transformed bovine 4-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (wild type) are used as detection objects. The transferred Oct4, Sox2, Klf4 and cMyc genes belong to the cattle gene, so that whether the cattle four-factor Oct4, Sox2, Klf4 and cMyc genes and the transgenic copy number thereof are transferred can be directly detected by adding a foreign vector gene sequence during the design of detection primers. The invention adopts the standard curve method fluorescent quantitative PCR to make the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic copy number standard curve. Respectively detecting transferred bovine four-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (transgenosis type) and non-transferred bovine 4-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells (wild type), and directly detecting whether a sample to be detected is transferred into bovine 4-factor Oct4, Sox2, Klf4 and cMyc genes and the transgenic copy number thereof. Lays a foundation for genetic stability screening experiments of transgenic cattle.
The purpose of the invention is realized by the following technical scheme: a primer for identifying bovine transgene copy number by standard curve method fluorescent quantitative PCR comprises primer pairs SEQ ID NO.1 and SEQ ID NO.2 used in internal reference gene GAPDH fluorescent quantitative PCR reaction, genes Oct4, Sox2, Klf4 and cMyc fluorescent quantitative PCR reaction, wherein the primer design parameters are that the length of the primer is 18-25bp, the Tm55-65 ℃, the difference between the upstream primer Tm value and the downstream primer Tm value of each pair of detection primers is not more than 4 ℃, and the size of a product is 100-300 bp.
A method for identifying the bovine transgene copy number by standard curve fluorescent quantitative PCR comprises the following steps:
(1) extracting genome DNA of transgenic bovine body cells, namely transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc and wild non-transformed bovine 4-factor Oct4, Sox2, Klf4 and cMyc;
(2) extracting the constructed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA;
(3) determining the quality and concentration of the genomic DNA of the bovine somatic cell samples of the transformed bovine four-factor and the non-transformed bovine 4-factor, and uniformly diluting the genomic DNA into 50ng/ul of the same concentration;
(4) designing and synthesizing a primer pair SEQ ID NO.1 and SEQ ID NO.2 according to a reference gene GAPDH DNA gene segment; designing and synthesizing a primer pair SEQ ID NO. 3-SEQ ID NO.10 according to the gene segments of the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA to be detected;
(5) fluorescence quantitative PCR by a standard curve method;
(6) dissolution curve analysis determined its uniqueness: if the dissolution curve is a single peak, the non-specific amplification does not occur in the fluorescent quantitative PCR amplification process, and therefore the data obtained by the fluorescent quantitative PCR amplification is reliable;
(7) the fluorescence quantitative PCR data was analyzed to determine the transgene copy number N.
The step (5) comprises the following steps:
(1) determining the quality and concentration of the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA, uniformly diluting the extracted bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA by 10x for 5 gradients which are respectively 10x0,10-1,10-2,10-3,10-4Doubling, respectively taking transgenic plasmid DNA with different dilutions as standard templates;
(2) using the genes to be detected Oct4, Sox2, Klf4, cMyc primers SEQ ID NO. 3-SEQ ID NO.10 and reference gene GAPDH primers SEQ ID NO.1 and SEQ ID NO.2 to 100,10-1,10-2,10-3,10-4Carrying out fluorescent quantitative PCR reaction on transgenic plasmid DNA standard substance templates with different dilutions, and drawing a log corresponding to a delta CT value according to the detection result of the fluorescent quantitative PCR reaction experiment of the transgenic plasmid DNA standard substance2 N(N represents copy number) of the following formula: log (log)2 NA × Δ CT + B N represents copy number, Δ CT ═ CT (gene to be measured) -CT (gapdh);
(3) and performing fluorescent quantitative PCR reaction on genomic DNA of bovine somatic cells with transferred bovine four-factor and bovine 4-factor samples by using genes to be detected Oct4, Sox2, Klf4, cMyc primers SEQ ID NO. 3-SEQ ID NO.10 and reference genes GAPDH primers SEQ ID NO.1 and SEQ ID NO.2, and obtaining a delta CT value, wherein the delta CT (transgenic type) is CT (transgenic type gene to be detected) -CT (transgenic type GAPDH) and the delta CT (wild type) is CT (wild type gene to be detected) -CT (wild type GAPDH) according to the detection result of the fluorescent quantitative PCR reaction experiment.
The step (7) comprises the following steps:
(1) according to the transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic bovine somatic cell delta CT (transgenic type) values, substituting the transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic bovine somatic cell delta CT (transgenic type) values into corresponding standard curves to perform copy number analysis;
(2) copy number analysis was performed by substituting the values of the untransformed bovine-derived four-factor Oct4, Sox2, Klf4, and cMyc bovine somatic Δ CT (wild-type) into the corresponding standard curve.
The invention has the advantages and beneficial effects that:
1. the invention can rapidly and accurately identify the copy number of the bovine exogenous gene by using the pair of detection primers.
2. The invention can rapidly identify the copy number of the bovine transgene by using the detection primer and the method, improves the detection efficiency of detecting the copy number of the transgenic animal transgene and saves the DNA dosage.
3. The detection primer and the detection method have the advantages of small amount of required experimental materials, small workload, time saving and accurate and reliable result, and have important theoretical and practical significance for guiding the identification of the bovine transgenic copy number, especially the detection of the transgenic copy number which is the same as the endogenous gene.
Drawings
FIG. 1 is a flow chart of the construction of the plasmid of the present invention.
FIG. 2 is a standard curve diagram of the inventive transgene copy number versus Δ CT values.
FIG. 3 is a graph of a dissolution curve analysis of the present invention.
Detailed Description
The invention is described in further detail below with reference to the following figures and detailed description:
designing a primer: GAPDH gene is used as an internal reference gene, and the accession number of the GAPDH gene in genbank is NM-001034034.2. The accession numbers of the bovine-derived four-factor Oct4, Sox2, Klf4 and cMyc genes in genbank are NM-174580.3, NM-001105463.2, NM-001105385.1 and NM-001046074.2 respectively. A gene amplification primer pair for identifying the bovine transgenic copy number by standard curve method fluorescent quantitative PCR (polymerase chain reaction), wherein primer premier 5.0 software is adopted to design internal reference gene GAPDH and fluorescent quantitative primers of Oct4, Sox2, Klf4 and cMyc to be detected, the length of the primers is 17-25bp, the difference between an upstream primer and a downstream primer cannot be too large, the length of a fluorescent quantitative product is 80-150bp, and the longest length is 300 bp; the Tm value is between 55 and 65 ℃, wherein the difference between the Tm value of the upstream primer and the Tm value of the downstream primer of each pair of detection primers is not more than 4 ℃; the product cannot form secondary structures.
The primer pair is further preferably: GAPDH, Oct4, Sox2, Klf4 and cMyc forward primer sequences, and reverse primer sequences are shown in a primer sequence table 1.
TABLE 1 primer sequence Listing
Transgenic plasmid: bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid construction method PiggyBac (PB) transposon + Oct4, Sox2, Klf4 and cMyc genes, and the construction sequence of the plasmid vector is shown in figure 1. The accession numbers of the bovine-derived four-factor Oct4, Sox2, Klf4 and cMyc genes in genbank are NM-174580.3, NM-001105463.2, NM-001105385.1 and NM-001046074.2 respectively. The construction of the vector adopts transposon PiggyBac to carry Oct4, Sox2, Klf4 and cMyc nuclear transcription factor, and PiggyBac is a transposon with higher transposition efficiency, can be accurately cut out and transposed in a plurality of species at higher frequency, and has simple operation and similar efficiency compared with other vectors, thus being a relatively ideal vector.
The construction method comprises the following steps: design Oct4, Sox2, Klf4 and cMyc cDNA amplification primers containing enzyme cutting sites EcolRI (upstream) and XbaI (downstream), amplify Oct4, Sox2, Klf4 and cMyc cDNA from bovine blastocysts by adopting the primers through a PCR method, purify 4 amplification products by using a PCR purification kit, then enzyme-cut PiggyBac transposon plasmids by adopting EcolRI and XbaI, recover a skeleton part, finally connect the skeleton and the PCR amplification products respectively for 16 degrees overnight, convert the connection products into Escherichia coli DH5 alpha, preliminarily identify positive clones by a colony PCR method, respectively pick 3 positive plasmids for further amplification, send a sequencing company to identify whether the plasmid is successfully constructed, and show that the sequencing result is successful to be used for subsequent transgenic experiments.
TABLE 2 plasmid construction Using primers Table
The method for identifying the bovine transgene copy number by standard curve fluorescent quantitative PCR comprises the following steps:
(1) extracting genomic DNA of transferred bovine four-factor Oct4, Sox2, Klf4, cMyc bovine somatic cell (transgenic type) and non-transferred bovine 4-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cell (wild type);
(2) the complete bovine four-factor Oct4, Sox2, Klf4, cMyc transgenic plasmid DNA has been constructed;
(3) determining the quality and concentration of the genomic DNA of the bovine somatic cells (transgenic type) with the transferred bovine four factors and the bovine somatic cells (wild type) with the non-transferred bovine 4 factors, and uniformly diluting the genomic DNA into 50ng/ul of the same concentration;
(4) designing and synthesizing primers according to the DNA gene segments of the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmids to be detected, wherein the design parameters are that the length of the primers is 18-25bp, the Tm is 55-65 ℃, the difference between the Tm values of the front primer and the rear primer is not more than 2 ℃, and the size of the product is 100-300 bp;
(5) performing fluorescent quantitative PCR by a standard curve method on a Thermo TCR0096 model fluorescent quantitative instrument:
(A) and determining the quality and concentration of the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA, uniformly diluting the extracted bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA by 10x for 5 gradients, wherein the 10 gradients are respectively 100,10-1,10-2,10-3,10-4Doubling, respectively taking transgenic plasmid DNA with different dilutions as standard templates;
(B) using the Oct4, Sox2, Klf4, cMyc primer and reference gene GAPDH primer pairs 100,10-1,10-2,10-3,10-4Carrying out fluorescent quantitative PCR reaction on transgenic plasmid DNA standard substance templates with different dilutions, and drawing a log corresponding to a delta CT value according to the detection result of the fluorescent quantitative PCR reaction experiment of the transgenic plasmid DNA standard substance2 N(N represents the number of copies)) The calculation formula of the standard curve is as follows: log (log)2 NA Δ CT + B N represents copy number, Δ CT ═ CT (gene to be tested) -CT (gapdh), and a standard curve of transgenic copy number versus Δ CT value is shown in fig. 2;
(C) performing fluorescent quantitative PCR reaction on genomic DNA of bovine somatic cells (transgenic type) with bovine four factors transferred and bovine 4 factors not transferred by using genes to be detected Oct4, Sox2, Klf4, cMyc primers and an internal reference gene GAPDH primer, and obtaining a delta CT value, a delta CT (transgenic type) value, a CT (transgenic type gene to be detected) -CT (transgenic type GAPDH) value and a delta CT (wild type gene to be detected) -CT (wild type GAPDH) value according to the experimental detection result of the fluorescent quantitative PCR reaction;
(6) dissolution curve analysis determined its uniqueness: if the dissolution curve is a single peak, it indicates that no non-specific amplification occurs during the fluorescent quantitative PCR amplification process, and therefore it is concluded that the data obtained by the fluorescent quantitative PCR amplification is reliable. The dissolution curve analysis is shown in FIG. 3;
(7) analyzing fluorescence quantitative PCR data, determining transgene copy number N:
(A) the bovine four-factor Oct4, Sox2, Klf4 and cMyc standard curve is prepared, and the calculation formula is as follows: log (log)2 NLog corresponding to A × Delta CT + B Delta CT value2 NThe standard curve of (N represents copy number), N represents copy number, Δ CT ═ CT (gene to be tested) -CT (gapdh), bovine four-factor Oct4, Sox2, Klf4, cMyc transgene copy number N corresponds to Δ CT value standard curve as shown in fig. 2;
(B) according to the transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic bovine somatic cell (transgenic) delta CT (transgenic) values, substituting the transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic bovine somatic cell (transgenic) delta CT (transgenic) values into a corresponding standard curve formula for copy number analysis;
(C) and (4) carrying out copy number analysis by substituting the values of untransformed bovine four factors Oct4, Sox2, Klf4 and cMyc bovine somatic cell (wild type) delta CT (wild type) into the corresponding standard curve formula.
Example analysis:
standard curve method fluorescent quantitative PCR identification of No.1 sample, No.2 sample and No.3 sample of bovine somatic cell (transgenic type) for Oct4 gene copy number analysis:
(1) the standard curve of Oct4 transgene copy number N versus Δ CT value is shown in FIG. 2: log (log)2 N＝-1.103*△ CT-2.952
(2) Carrying out fluorescent quantitative PCR reaction on bovine somatic cell No.1 sample genomic DNA and bovine somatic cell No.2 sample genomic DNA of the transferred bovine source Oct4 gene by using a to-be-detected gene Oct4 primer and an internal reference gene GAPDH primer, obtaining a Delta CT value according to the detection result of a fluorescent quantitative PCR reaction experiment, and calculating the result according to the formula of Delta CT (transgenic type) CT (transgenic type to-be-detected Oct4 gene) -CT (transgenic type GAPDH), wherein the Delta CT (transgenic type No.1 sample) is 14.71-18.04-3.33 Delta CT (transgenic type No.2 sample) is 15.00-18.55-3.55;
(3) and performing fluorescent quantitative PCR reaction on genomic DNA of a bovine somatic cell No.3 sample of the untransformed bovine source Oct4 gene by using an Oct4 primer of the gene to be detected and a GAPDH primer of the reference gene, obtaining a Delta CT value according to the detection result of the fluorescent quantitative PCR reaction experiment, and calculating the Delta CT (wild type) according to the formula of the Delta CT (wild type Oct4 gene to be detected) -CT (wild type GAPDH), wherein the Delta CT (wild type No.3 sample) is 16.11-17.25-1.41
(4) Δ CT (transgene type No.1 sample) — 3.33, Δ CT (transgene type No.2 sample) — 3.55, and Δ CT (wild type No.3 sample) — 1.41. Substituting into Oct4 transgene copy number N corresponding to Delta CT value standard curve formula log2 NThe copy number (sample No.1 transgenic) was 1.65, the copy number (sample No.2 transgenic) was 1.95, and the copy number (sample No.3 wild) was 0.38, calculated from-1.103 × Δ CT-2.952.
(5) And analyzing according to the calculation result of the transgenic copy number, wherein the transgenic copy number of the transgenic No.1 sample is 2, the transgenic copy number of the transgenic No.2 sample is 2, and the transgenic copy number of the wild No.3 sample is 0. The detection primer and the detection method have the advantages of small amount of required experimental materials, small workload, time saving and accurate and reliable result, and have important theoretical and practical significance for guiding the identification of the bovine transgenic copy number, especially the detection of the transgenic copy number which is the same as the endogenous gene.
The invention adopts an SYBR Green II real-time fluorescent quantitative PCR method, which is to add SYBR Green II fluorescent dye into a PCR reaction system, wherein the dye can be combined with all DNA double helix minor groove regions and has the capability of generating Green fluorescence by excitation; when the fluorescent probe is combined with double-stranded DNA synthesized by PCR, fluorescence is generated under the irradiation of exciting light, and the increase of the fluorescence signal intensity is completely synchronous with the increase of a PCR product, so that PCR amplification data can be indirectly obtained by detecting the fluorescence signal intensity in the PCR reaction process in real time; the transgene copy number can be directly obtained by analysis through a standard curve fluorescent quantitative PCR method.
In recent years, the detection of transgene copy number by high-throughput, rapid and sensitive quantitative PCR methods is becoming popular among researchers. The detection objects are the transgenic bovine four-factor Oct4, Sox2, Klf4 and cMyc bovine somatic cells which are constructed in the laboratory. The transferred Oct4, Sox2, Klf4 and cMyc genes belong to cattle, and a foreign vector gene is added during the design of detection primers, so that whether the Oct4, Sox2, Klf4 and cMyc foreign genes are transferred or not and the copy number of the genes can be directly detected.
The above-mentioned embodiments are only for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to carry out the same, and the present invention shall not be limited to the embodiments, i.e. the equivalent changes or modifications made within the spirit of the present invention shall fall within the scope of the present invention.
Sequence listing
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Claims (4)
1. A primer for identifying bovine transgene copy number by standard curve method fluorescent quantitative PCR is characterized by comprising primer pairs SEQ ID NO.1 and SEQ ID NO.2 used in internal reference gene GAPDH fluorescent quantitative PCR reaction, genes to be detected Oct4, Sox2, Klf4 and primer pairs SEQ ID NO. 3-SEQ ID NO.10 used in cMyc fluorescent quantitative PCR reaction, wherein the primer design parameters are that the length of the primer is 18-25bp, the Tm is 55-65 ℃, the difference between the upstream primer Tm value and the downstream primer Tm value of each pair of detection primers is not more than 4 ℃, and the product size is 100-300 bp.
2. A method for identifying the bovine transgene copy number by standard curve fluorescent quantitative PCR is characterized by comprising the following steps:
(1) extracting genome DNA of transgenic bovine body cells, namely transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc and wild non-transformed bovine 4-factor Oct4, Sox2, Klf4 and cMyc;
(2) extracting the constructed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA;
(3) determining the quality and concentration of the genomic DNA of the bovine somatic cell samples of the transformed bovine four-factor and the non-transformed bovine 4-factor, and uniformly diluting the genomic DNA into 50ng/ul of the same concentration;
(4) designing and synthesizing a primer pair SEQ ID NO.1 and SEQ ID NO.2 according to a reference gene GAPDH DNA gene segment; designing and synthesizing a primer pair SEQ ID NO. 3-SEQ ID NO.10 according to the gene segments of the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA to be detected;
(5) fluorescence quantitative PCR by a standard curve method;
(6) dissolution curve analysis determined its uniqueness: if the dissolution curve is a single peak, the non-specific amplification does not occur in the fluorescent quantitative PCR amplification process, and therefore the data obtained by the fluorescent quantitative PCR amplification is reliable;
(7) the fluorescence quantitative PCR data was analyzed to determine the transgene copy number N.
3. The method for identifying the bovine transgene copy number by standard curve fluorescent quantitative PCR (polymerase chain reaction) according to claim 2, wherein the step (5) comprises the following steps:
(1) determining the quality and concentration of the bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA, uniformly diluting the extracted bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic plasmid DNA by 10x for 5 gradients which are respectively 10x0,10-1,10-2,10-3,10-4Doubling, respectively taking transgenic plasmid DNA with different dilutions as standard templates;
(2) by usingOct4, Sox2, Klf4, cMyc primer SEQ ID NO. 3-SEQ ID NO.10 to be tested, and reference gene GAPDH primer SEQ ID NO.1 and SEQ ID NO.2 pair 100,10-1,10-2,10-3,10-4Carrying out fluorescent quantitative PCR reaction on transgenic plasmid DNA standard substance templates with different dilutions, and drawing a log corresponding to a delta CT value according to the detection result of the fluorescent quantitative PCR reaction experiment of the transgenic plasmid DNA standard substance2 N(N represents copy number) of the following formula: log (log)2 NA × Δ CT + B N represents copy number, Δ CT ═ CT (gene to be measured) -CT (gapdh);
(3) and performing fluorescent quantitative PCR reaction on genomic DNA of bovine somatic cells with transferred bovine four-factor and bovine 4-factor samples by using genes to be detected Oct4, Sox2, Klf4, cMyc primers SEQ ID NO. 3-SEQ ID NO.10 and reference genes GAPDH primers SEQ ID NO.1 and SEQ ID NO.2, and obtaining a delta CT value, wherein the delta CT (transgenic type) is CT (transgenic type gene to be detected) -CT (transgenic type GAPDH) and the delta CT (wild type) is CT (wild type gene to be detected) -CT (wild type GAPDH) according to the detection result of the fluorescent quantitative PCR reaction experiment.
4. The method for identifying the bovine transgene copy number by standard curve fluorescent quantitative PCR (polymerase chain reaction) according to claim 2, wherein the step (7) comprises the following steps:
(1) according to the transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic bovine somatic cell delta CT (transgenic type) values, substituting the transformed bovine four-factor Oct4, Sox2, Klf4 and cMyc transgenic bovine somatic cell delta CT (transgenic type) values into corresponding standard curves to perform copy number analysis;
(2) copy number analysis was performed by substituting the values of the untransformed bovine-derived four-factor Oct4, Sox2, Klf4, and cMyc bovine somatic Δ CT (wild-type) into the corresponding standard curve.
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| WO2011071936A2 (en) * | 2009-12-07 | 2011-06-16 | Gary Dahl | Compositions and methods for reprogramming eukaryotic cells |
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| CN107988329A (en) * | 2017-11-13 | 2018-05-04 | 广东艾时代生物科技有限责任公司 | It is a kind of to be used to identify that multipotential stem cell is endogenous and detection primer group, kit and the detection method of external source versatility gene expression |
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| WO2011071936A2 (en) * | 2009-12-07 | 2011-06-16 | Gary Dahl | Compositions and methods for reprogramming eukaryotic cells |
| CN102747104A (en) * | 2011-04-21 | 2012-10-24 | 中国科学院上海生命科学研究院 | Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method |
| CN107988329A (en) * | 2017-11-13 | 2018-05-04 | 广东艾时代生物科技有限责任公司 | It is a kind of to be used to identify that multipotential stem cell is endogenous and detection primer group, kit and the detection method of external source versatility gene expression |
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