CN116479096A

CN116479096A - Universal standard for determining genome iCre transgene copy number based on absolute quantitative PCR, preparation and detection method thereof

Info

Publication number: CN116479096A
Application number: CN202310366263.3A
Authority: CN
Inventors: 刁戈; 韩健; 郭建新; 田敏; 黄洁; 李润博
Original assignee: Chinese Peoples Liberation Army Army Specialized Medical Center
Current assignee: Chinese Peoples Liberation Army Army Specialized Medical Center
Priority date: 2023-04-07
Filing date: 2023-04-07
Publication date: 2023-07-25

Abstract

The invention discloses a universal standard for determining genome iCre transgene copy number based on absolute quantitative PCR, a preparation method and a determination method thereof, wherein a large primer PCR method (megaprimer PCR) is adopted to construct a standard with the accurate Copy Number Ratio (CNR) of a target gene and a control gene. Based on the standard, an absolute quantitative PCR method is adopted to accurately detect the copy number of the improved Cre (Cre) gene in the genome. The standard was suitable for all model animals that underwent the cre transgene. The standard can be used for rapidly and accurately detecting and quantifying the copy number of the iCre transgene in the genome of the individual, and is greatly helpful for the establishment and application of the animal with the iCre transgene mode.

Description

Universal standard for determining genome iCre transgene copy number based on absolute quantitative PCR, preparation and detection method thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a universal standard for determining genome iCre transgene copy number based on absolute quantitative PCR, and a preparation and detection method thereof.

Background

The Cre-loxP recombination system, which is to locate a specific DNA sequence and splice the DNA sequence by Cre recombinase, consists of two parts of Cre recombinase and loxP site, and can control the gene expression spatially and temporally. Cre recombinase is a type I topoisomerase derived from E.coli phage P1, and is also a tyrosine recombinase which recognizes loxP sites and catalyzes recombination of DNA between the loxP sites. The Cre-loxP recombination system has now become a common tool for creating animals with conditional expression patterns of genes. Among them, the construction of a model animal of Cre recombinase expression is one of the keys. One common approach is to randomly integrate the exogenous specific promoter with the Cre recombinase gene sequence into the genome of the animal using prokaryotic microinjection. The method can rapidly obtain the required animal model, and has low cost. However, due to the randomness of the integration site and the uncontrolled gene copy number, the expression of Cre recombinase is likely to be affected. In order to obtain an animal individual stably expressing Cre recombinase, the resulting transgenic animal should be bred for several generations to obtain a single copy of the Cre gene. Thus, there is a need to establish an efficient and accurate method for detecting the Cre copy number integrated in the genome of transgenic animals. The Cre (modified Cre recombinase) is an optimized form of Cre recombinase suitable for use in mammalian systems.

Fluorescent quantitative PCR is a common method for detecting gene copy number variation, and can be carried out in most laboratories. It is divided into two methods, absolute quantification and relative quantification. The relative quantification method requires designing primers with the same amplification efficiency of the target gene and the control gene, otherwise, the result has larger error. The difficulty with absolute quantification is the lack of a standard with an accurate copy number ratio of the gene of interest to the control gene.

Disclosure of Invention

The invention constructs a standard substance with the accurate copy number ratio (copy number ratio, CNR) of a target gene and a control gene by adopting a large primer PCR method (megaprimerPCR). Based on the standard, an absolute quantitative PCR method is adopted to accurately detect the copy number of the improved Cre (Cre) gene in the genome. The standard was suitable for all model animals that underwent the cre transgene. Based on the above, the invention provides the following technical scheme:

a preparation method of a universal standard for determining genome iCre transgene copy number based on absolute quantitative PCR comprises the following steps:

1) Taking plasmid containing the ORF complete sequence of the iCre gene as a template, adopting Megaprimer PCR to carry out first-round PCR amplification, and separating the copy number of the iCre gene from the obtained PCR product to be n ₁ Wherein said n ₁ =1, 2 or n ₁ ＝1,2,4；

2) The copy number of the iCre gene obtained by the first round of PCR amplification is n ₁ The fragment of (2) is connected into pUCm-T vector to obtain recombinant plasmid 1, the recombinant plasmid cloned with 2 XiCre fragment or 4 XiCre fragment is used as DNA template for second amplification of megaprimer PCR reaction to make second amplification, and the copy number of iCre gene is separated from the obtained PCR product ₂ Wherein said n ₂ Including any one of an even number of 4 or more and 32 or less; n obtained by separation ₂ The x iCre tandem repeat fragment is also ligated into pUCm-T vector to obtain recombinant plasmid 2;

3) Respectively amplifying 1 copy of IL-2 gene fragment 1 containing EcoRI and KpnI enzyme cutting sites and 1 copy of IL-2 gene fragment 2 containing BamHI and XbaI sites by taking DNA of a species to be detected of the universal standard substance as a template;

4) Taking the product obtained in step 2) and containing n ₁ ×iCre、n ₂ pUCm-T recombinant plasmids 1 and 2 of the XiCre gene fragment, respectively, into which IL-2 gene fragment 1 is constructed by means of enzyme digestion connection, so as to obtain the iCre/IL-2 with CNR of n respectively ₁ 、n ₂ Recombinant plasmids 3 and 4 of (a);

in addition, taking recombinant plasmid 1 with the CNR of the iCre/IL-2 as 1, and constructing 1 copy of IL-2 gene fragment 2 into the recombinant plasmid sequence in an enzyme digestion connection mode to obtain recombinant plasmid with the CNR of the iCre/IL-2 as 0.5;

thus, the iCre/IL-2 CNR was obtained as 0.5 and n, respectively ₁ 、n ₂ Is a universal plasmid vector standard of (a).

In the technical scheme of the preparation method, the n is as follows ₂ Is an integer multiple of 4, preferably the n ₂ =4, 8,16 or n ₂ =4, 8,16,32 or n ₂ =8, 16 or n ₂ = 8,16,32; preferably n ₁ ＝1,2,4，n ₂ ＝8,16。

In the technical scheme of the preparation method, the primers used for PCR amplification in the steps 1) and 2) are MegP-F:5' -TGAACGCCACTGACTTTGAC-3, megP-R:5'-CTTGTCAAAGTCAGTGCGTTCAGCAGCCACACCATTCTTTCT-3';

the reaction conditions for the first round of PCR amplification are: 3min at 95 ℃;98℃15sec,50℃30sec,68℃1min,25 cycles; 15 cycles of 98℃15sec,62℃30sec,68℃1 min;

the reaction conditions for the second round of PCR amplification are: 3min at 95 ℃;98℃15sec,62℃30sec,68℃3min,35 cycles.

In the technical scheme of the preparation method, the PCR product direct TA connection method is adopted in the step 2), and the copy number of the iCre gene is n ₁ 、n ₂ Adding A at two ends of the PCR product, and connecting the product obtained by the reaction of adding A into a pUCm-T vector through TA cloning;

in the step 4), the recombinant plasmids 1 and 2 and the IL-2 gene fragment 1 are respectively subjected to restriction enzyme digestion by EcoRI and KpnI and then are connected by T4 ligase; the recombinant plasmid 1 and 1 copy of IL-2 gene fragment 2 were digested with BamHI and XbaI restriction enzymes, respectively, and then ligated using T4 ligase.

In the technical scheme of the preparation method, the organism to be detected is a mouse, in the step 3), the sequences of the amplification primer pairs of the IL-2 gene fragment 1 are shown as SEQ ID NO.5 and SEQ ID NO.6, and the sequences of the amplification primer pairs of the IL-2 gene fragment 2 are shown as SEQ ID NO.7 and SEQ ID NO. 8.

The invention also provides a universal standard prepared by the preparation method of any one of the above.

The invention also provides a method for determining the copy number of the iCre transgene based on an absolute quantitative PCR method, which adopts the universal standard to determine, and comprises the following steps:

1) Establishing a standard curve:

taking a plurality of standard products, wherein the standard products at least comprise standard products with the CNR of iCre/IL-2 of 0.5 and 16 respectively or standard products with the CNR of iCre/IL-2 of 0.5 and 32 respectively;

respectively performing fluorescent quantitative PCR detection by using standard substance as template and respectively adopting amplification primers of iCre gene and IL-2 gene, respectively reading Ct _{Target gene} And Ct _{Control genes} Delta Ct was calculated for each standard sample:

ΔCt＝Ct _{target gene} -Ct _{Control genes} The (I)

Log of standard substance ₂ (CNR) is an abscissa, delta Ct is an ordinate, and a standard curve is drawn to obtain a standard curve regression equation formula:

y ₁ ＝ax ₁ +b, formula (II)

In the formula (II), x ₁ Is log of ₂ (CNR) value (i.e. cnr=2 ^x1 )，y ₁ Delta Ct value;

3) Detecting a sample to be detected:

extracting genome DNA of a biological sample to be detected containing the iCre gene; respectively performing fluorescent quantitative PCR detection by using the extracted genome DNA as a template and adopting amplification primers of the iCre gene and the IL-2 gene, and respectively reading Ct of a sample to be detected _{Target gene} And Ct _{Control genes} Calculating the delta Ct of the sample to be detected according to the formula (I), substituting the calculated delta Ct into the formula (II) to obtain the x of the sample to be detected ₁ According to the formula (III), calculating the actual copy number of the iCre gene in the sample species to be detected, wherein the specific calculation formula is as follows:

y ₂ ＝x ₂ *2 ^x1 the (III)

In the formula (III), y ₂ For the actual copy number, x, of the cre gene in the genome of the sample organism to be tested ₂ Chromosome of organism to which sample to be measured belongsMultiple.

In the technical scheme of the method for determining the copy number of the cre transgene based on the absolute quantitative PCR method, a quality control sample is used when a sample to be detected is detected, the quality control sample comprises a quality control product A and/or a quality control product B, the quality control product A is an individual sample of the cre transgene mouse with the copy number of the cre gene being 1 through whole genome sequencing, and the quality control product B is a plasmid sample with the CNR of the cre/IL-2 being 1.

In the technical scheme of the method for determining the copy number of the iCre transgene based on the absolute quantitative PCR method, the species to be detected is a mouse, the fluorescent quantitative PCR amplification primer sequences of the iCre gene are shown as SEQ ID NO.9 and SEQ ID NO.10, and the fluorescent quantitative PCR amplification primer sequences of the IL-2 gene are shown as SEQ ID NO.11 and SEQ ID NO. 12;

the reaction system is as follows: 2X QuantiNova SYBR Green PCR Master mix. Mu.l each of forward and reverse primers 0.5. Mu.l each, 1. Mu.l of DNA template, ddH ₂ O was made up to 20. Mu.l;

the PCR reaction conditions were: 3min at 95 ℃;98℃for 5sec,60℃for 10sec,40 cycles.

In the method of any one of the above, the ORF sequence of the iCre gene is shown as SEQ ID NO.1, and the IL-2 gene amplification sequence is shown as SEQ ID NO. 4;

the standard comprises plasmid vector standards with iCre/IL-2 CNR of 0.5, 1,2, 4,8, 16.

When a standard curve is constructed, because the copy number of the cre in the standard substance needs to be increased in multiple ratio, and the tandem repeat fragments exceeding a certain length are difficult to successfully obtain at present, the cre gene tandem repeat fragments containing 2 or 4 (but not 3) cre genes are obtained by adopting the first PCR amplification of the megaprimer PCR reaction, and the recombinant plasmid constructed by the fragments is used as a template for carrying out the second PCR amplification to obtain the cre gene tandem repeat fragments (such as 8X and 16X) containing even multiple relation; if the recombinant plasmid constructed by 3 iCre gene tandem repeats obtained by the first PCR amplification is used as a template for the second PCR amplification, 9×, 27×, 81× iCre gene tandem repeats are theoretically obtained, but since the 27×, 81× iCre gene tandem repeats are too long to successfully obtain a recombinant plasmid truly comprising 27 or 81 tandem repeats, the present application selects a recombinant plasmid constructed by 2 or 4 iCre gene tandem repeats as a template for the second PCR amplification.

The method is suitable for constructing standard products for detecting the copy number of the iCre transgene in animals in different modes and detecting the copy number of the iCre transgene in the standard products, and is not limited to the experimental object mice in the embodiment. When the organism to be tested is an animal of other modes, then the biological sample is obtained according to y ₂ ＝x ₂ *2 ^x1 The actual copy number of the cre gene therein is calculated according to formula (III), e.g.y for triploid organisms ₂ ＝3*2 ^x1 。

Among them, the control gene selects IL-2, and the IL-2 gene is a gene which most of high-grade organisms have, and the gene has stable structure, and the copy number of the gene is constant in organisms, such as 2 copies in mice, so that the gene is selected as the control gene in a standard plasmid. According to the species of the sample to be tested, the IL-2 gene of the species is selected as a control gene.

When a standard curve is constructed, the selected standard at least contains a standard with the CNR of iCre/IL-2 of 0.5, 16 or 32, and the CNR value is near 0.5 when the gene copy number is 1, so that the standard needs to contain the standard with the CNR of 0.5 of the iCre/IL-2; the standard product containing the iCre/IL-2 with the CNR of 16 or 32 ensures that the CNR value of the sample to be detected is within the range of the standard curve so as to ensure the detection accuracy (normally, the CNR of the iCre/IL-2 of the sample to be detected is not more than 16); in order to ensure accuracy, a plurality of standard substances are selected to construct a standard curve.

The beneficial effects of the invention are as follows: the standard product for detecting the copy number of the iCre transgene in the genome constructed by the invention is suitable for all model animals which adopt a random transgene method to express the iCre gene. The standard can be used for rapidly and accurately detecting and quantifying the copy number of the iCre transgene in the genome of the individual, and is greatly helpful for the establishment and application of the animal with the iCre transgene mode.

Drawings

FIG. 1 shows the results of product identification obtained after the first round of megaprimer PCR amplification, M: DNAmarker, DL5000. FIG. 2 shows the product identification results obtained after the second round of megaprimer PCR amplification, M: DNAmarker, DNALadder (0.1-10 kb).

FIG. 3 is a schematic diagram of the structure of a standard plasmid vector with iCre/IL-2 CNR of 1,2, 4,8, 16.

FIG. 4 is a schematic diagram of the structure of a standard plasmid vector with a CNR of iCre/IL-2 of 0.5.

FIG. 5 is a standard graph obtained in example 3.

FIG. 6 is a schematic structural diagram of a plasmid vector of quality control B.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified; the chemicals, biological materials and reagents used, unless otherwise specified, are all conventional in the art and are commercially available.

EXAMPLE 1 construction of the iCre tandem repeat

The principle is as follows: the ORF (open reading frame ) sequence of the cre gene, which is 336bp in length, was constructed as a different number of tandem repeats by two rounds of megaprimer PCR reactions. The obtained tandem repeat sequence was ligated into pUCm-T vector by TA cloning to obtain plasmid vectors containing 1,2, 4,8 and 16 iCre gene tandem repeat fragments, respectively. The IL-2 (interleukin 2) gene fragment of 191bp in length, which is the control gene, was cloned into the plasmid vector containing the cre gene fragment by means of cleavage. Thus, plasmid vectors were obtained in which the copy number ratios (copy number ratio, CNR) of iCre/IL-2 were 1,2, 4,8 and 16, respectively. Then, in the plasmid vector with the iCre/IL-2 CNR of 1, cloning the IL-2 sequence of the other fragment into the vector sequence through other enzyme cutting sites to obtain the plasmid vector with the iCre/IL-2 CNR of 0.5. Through the mode, the standard products with the iCre/IL-2 CNR of 0.5, 1,2, 4,8 and 16 are prepared.

In each quantitative PCR reaction, the standard was amplified using the standard as a template, and a standard curve was drawn. And then carrying the corresponding value obtained by amplifying the unknown sample to be detected into a standard curve for calculation, thus obtaining the specific copy number of the iCre gene in the sample.

The iCre gene ORF sequence is as follows (SEQ ID NO. 1):

5’-TGAACGCACTGACTTTGACCAAGTCAGATCCCTGATGGAGAACTCTGACAGATGCCAGGACATCAGGAACCTGGCCTTCCTGGGCATTGCCTACAACACCCTGCTGCGCATTGCCGAAATTGCCAGAATCAGAGTGAAGGACATCTCCCGCACCGATGGTGGGAGAATGCTGATCCACATTGGCAGGACCAAGACCCTGGTGTCCACAGCTGGTGTGGAGAAGGCCCTGTCCCTGGGGGTTACCAAGCTGGTGGAGAGATGGATCTCTGTGTCTGGTGTGGCTGATGACCCCAACAACTACCTGTTCTGCCGGGTCAGAAAGAATGGTGTGGCTGC-3’。

(1) Megaprimer PCR first round amplification:

the primer is as follows:

the forward primer MegaP-F (SEQ ID NO. 2): 5'-TGAACGCCACTGACTTTGAC-3',

reverse primer MegaP-R (SEQ ID NO. 3):

5’-CTTGTCAAAGTCAGTGCGTTCAGCAGCCACACCATTCTTTCT-3’。

the reaction system is as follows:

component (A)	Volume (mul)
		ddH ₂ O	3.5
2X PCR Buffer for KOD FX Neo (Toyobo, japan)	10
		dNTP(2mM)	4
KOD FX Neo (5U/. Mu.l) (Toyobo, japan)	0.4
		Forward primer MegaP-F (10 pmol/. Mu.l)	0.1
Reverse primer MegaP-R (10 pmol/. Mu.l)	1
		DNA template (100 ng/. Mu.l)	1
Total volume of	20

The DNA template was a pcDNA3.1 plasmid containing the complete sequence of the ORF of the iCre gene, synthesized by Shanghai Biotechnology.

PCR reaction conditions:

first, the amplified product was identified by 1% agarose gel (FIG. 1).

Next, a direct TA ligation of PCR products was used to add A to both ends of the resulting PCR amplification product.

The reaction system:

component (A)	Volume (mul)
		10 XTaq Buffer (Biyun Tian, china)	6
dATP(50mM)	0.5
		Taq enzyme (5U/. Mu.l) (Biyun Tian, china)	1
PCR products	52.5
		Total volume of	60

Reaction conditions: 72℃for 30min.

After obtaining the product with a added to both ends of the sequence, 1× (i.e. 1 cre gene sequence), 2× (i.e. 2 tandem repeats) and 4× (i.e. 4 tandem repeats) cre tandem repeat fragments thereof can be separated by method one and purified for recovery to construct a plasmid vector.

The method comprises the following steps: the product from the addition A reaction was subjected to gel recovery and the kit was Gel Extraction Kit (Omega bio-tek, USA). Thus, 1×,2×, and 4×cre tandem repeat fragments were separated and recovered, and the resulting 1×,2×, and 4×cre tandem repeat fragments were ligated into pUCm-T vector (Shanghai Ind. Co.) by TA cloning, positive clones were taken, and DNA was verified by sequencing.

This step may also be performed using method two: the product from the addition A reaction was purified directly using the Cycle Pure Kit (Omega bio-tek, USA). The purified product was ligated into pUCm-T vector (Shanghai) by TA cloning, and the monoclonal was selected and positive clones containing 1×,2× and 4× iCre tandem repeats were identified by colony PCR, respectively. The corresponding positive clones were then taken to extract plasmids, pUCm-T vectors were obtained from which 1X, 2X and 4X iCre tandem repeats were cloned, respectively, and DNA was verified by sequencing.

pUCm-T vector cloned with 4 XiCre fragment was used as DNA template for the second round of amplification of megaprimer PCR reaction.

(2) Megaprimer PCR second round amplification:

the reaction system:

Reaction conditions:

after a second round of PCR amplification, the products were identified using 1% agarose gel (FIG. 2). The PCR amplification products contained 8X and 16X iCre fragments. Then adopting the same TA connection reaction, and adding A at two ends of the product. The resulting addition A product was isolated and cloned into pUCm-T vectors by the same manner as described above, and the pUCm-T vectors were finally obtained, in which the 8X and 16X iCre tandem repeats were cloned, and were verified by sequencing.

EXAMPLE 2 preparation of Standard plasmid

The purpose of this example was to prepare plasmid vector standards with a CNR of 0.5, 1,2, 4,8,16 for iCre/IL-2 using the plasmid vector containing the iCre tandem repeat sequence obtained in example 1.

(1) Preparation of control Gene IL-2 fragment

Mouse IL-2 gene amplified fragment sequence (SEQ ID NO. 4):

5’-CGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGTAAGGTGTTGCTTTATTTGCTAATCTGGAAATAAAATAGAGAAGAAATGCATTTTTAAGTGGCTTGTCATTTCTGGTCTTTGATGGGTTCTGTG-3’。

the PCR method is adopted, and primers with enzyme cutting sites are utilized to amplify IL-2 gene fragments with different enzyme cutting sites from wild animal DNA samples.

The primer is as follows:

* And (3) injection: the underlined bases are cleavage site fragments with a protecting base.

The reaction system is as follows:

component (A)	Volume (mul)
		ddH ₂ O	8
2 XTaq Master Mix (Vazyme, china)	10
		Forward primer IL-2-F-EcoRI/IL-2-F-BamHI (10 pmol/. Mu.l)	0.5
Reverse primer IL-2-R-KpnI/IL-2-R-XbaI (10 pmol/. Mu.l)	0.5
		DNA template (100 ng/. Mu.l)	1
Total volume of	20

PCR reaction conditions:

the resulting PCR amplification product was purified by a Cycle Pure Kit (Omega bio-tek, USA). (2) Construction of standard quality pellets with accurate copy number ratio

On the basis of the pUCm-T vector containing 1,2, 4,8 and 16×iCre gene fragments, respectively, a gene fragment of the control gene IL-2 containing EcoRI and KpnI cleavage sites was constructed therein by cleavage ligation.

Carrier enzyme cleavage reaction system:

component (A)	Volume (mul)
		ddH ₂ O	14
10X FastDigest Green Buffer (Thermo Scientific, U.S.)	2
		FastDiget EcoRI (Thermo Scientific, U.S.A.)	1
FastDiget KpnI (Thermo Scientific, U.S.A.)	1
		Vector DNA (not more than 1. Mu.g)	2
Total volume of	20

Fragment cleavage reaction system:

component (A)	Volume (mul)
		ddH ₂ O	16
10X FastDigest Green Buffer (Thermo Scientific, U.S.)	2
		FastDiget EcoRI (Thermo Scientific, U.S.A.)	1
FastDiget KpnI (Thermo Scientific, U.S.A.)	1
		DNA fragment (not more than 0.2. Mu.g)	10
Total volume of	30

Reaction conditions: 37℃for 30min.

After the cleavage reaction, the vector and fragment were purified using the Cycle Pure Kit (Omega bio-tek, U.S.A.), respectively.

The vector and fragment were ligated by T4 ligase, the ligated product was transformed, and positive clones were selected. And (3) connecting a reaction system:

component (A)	Volume (mul)
		ddH ₂ O	2.5
10X T4 DNA Ligase Buffer (Thermo Scientific, U.S.)	1
		T4 DNA Ligase (Thermo Scientific, U.S.A.)	0.5
Vector DNA (100 ng)	1
		DNA fragment (200 ng)	5
Total volume of	10

Reaction conditions: 4℃overnight.

Thus, a standard plasmid vector having iCre/IL-2 CNR of 1,2, 4,8,16, respectively, was obtained, and the schematic structure thereof is shown in FIG. 3.

Next, the gene fragment of the control gene IL-2 containing BamHI and XbaI cleavage sites was cloned into a plasmid vector having a CNR of 1 of iCre/IL-2 by cleavage to give a plasmid vector having a CNR of 0.5 of iCre/IL-2, the structure of which is schematically shown in FIG. 4. Wherein, the carrier enzyme digestion reaction system is as follows:

component (A)	Volume (mul)
		ddH ₂ O	14
10X FastDigest Green Buffer (Thermo Scientific, U.S.)	2
		FastDiget BamHI (Thermo Scientific, U.S.A.)	1
FastDiget XbaI (Thermo Scientific, U.S.A.)	1
		Vector DNA (not more than 1. Mu.g)	2
Total	20

The fragment enzyme digestion reaction system is as follows:

component (A)	Volume (mul)
		ddH ₂ O	16
10X FastDigest Green Buffer (Thermo Scientific, U.S.)	2
		FastDiget BamHI (Thermo Scientific, U.S.A.)	1
FastDiget XbaI (Thermo Scientific, U.S.A.)	1
		DNA fragment (not more than 0.2. Mu.g)	10
Total	30

Reaction conditions: 37℃for 30min.

And (3) connecting a reaction system:

component (A)	Volume (mul)
		ddH ₂ O	2.5
10X T4 DNA Ligase Buffer (Thermo Scientific, U.S.)	1
		T4 DNA Ligase (Thermo Scientific, U.S.A.)	0.5
Vector DNA (100 ng)	1
		DNA fragment (200 ng)	5
Total	10

Reaction conditions: 4℃overnight.

Through the mode, the plasmid vector standard product with the iCre/IL-2 CNR of 0.5 is prepared.

Thus, plasmid vector standards with iCre/IL-2 CNR of 0.5, 1,2, 4,8,16 were prepared.

Example 3 establishment and application of fluorescent quantitative PCR detection System

1. Establishment of fluorescent quantitative PCR detection system

(1) PCR reaction System and conditions

Primer information is as follows:

the reaction system is as follows:

the reaction conditions were as follows:

(2) Standard curve establishing and calculating method

A standard plasmid vector with the CNR of iCre/IL-2 of 0.5, 1,2, 4,8 and 16 prepared in example 2 is used as a DNA template for real-time fluorescence quantitative PCR, and the fluorescence quantitative PCR is carried out according to the reaction system and the conditions.

First, the delta Ct of each standard sample is calculated,

ΔCt＝Ct _{target gene} -Ct _{Control genes} The (I)

In the formula (I), the Ct value refers to the number of cycles undergone by the fluorescent signal in each reaction tube reaching a set threshold, ct _{Target gene} Refers to the Ct value, ct of the iCre gene in the detected standard plasmid vector _{Control genes} Refers to the Ct value of the IL-2 gene detected in the standard plasmid vector.

Log of standard substance ₂ (CNR) is an abscissa, delta Ct is an ordinate, and a standard curve of each time (due to errors caused by sample preparation, sample addition and other operations, the standard curve obtained by each detection has a certain difference) is drawn, so that a standard curve regression equation formula is obtained:

y ₁ ＝ax ₁ +b, formula (II)

In the formula (II), a represents a slope, and b represents an intercept.

Taking the standard curve obtained in the study of this embodiment as an example (as shown in fig. 5), a calculation formula is obtained:

y ₁ ＝-0.9917x ₁ +1.885，R ² ＝0.9952

in the formula (II), y ₁ Is delta Ct value, x ₁ Is log of ₂ (CNR) value (i.e. cnr=2 ^x1 )

For different model animals, according to the fact that the detected sample is several times of organisms, multiplying the samples by corresponding times to obtain the actual copy number of the iCre gene in the model animals, wherein the specific calculation formula is as follows:

y ₂ ＝x ₂ *2 ^x1 the (III)

In the formula (III), y ₂ For the actual copy number, x, of the cre gene in the genome of the sample organism to be tested ₂ To which the sample to be measured belongsChromosome fold of an organism.

For example, the mouse is a 2-fold organism, and thus the actual copy number of the cre gene in the mouse is twice the CNR value, i.e. the actual copy number y of the cre gene in the mouse ₂ ＝2*2 ^x1 。

When R of the standard curve is obtained ² When the value reaches more than 0.99, the standard curve meets the requirement, and the secondary detection result is effective.

For an unknown sample to be detected, obtaining Ct through fluorescent quantitative PCR _{Target gene} And Ct _{Control genes} Calculating according to formula (I) to obtain delta Ct, and substituting delta Ct value into formula (II) to obtain x ₁ Will be 2 ^x1 And substituting the actual copy number of the iCre gene in the biological sample to be detected into the formula (III) to calculate.

(3) Quality control sample

In order to ensure the accuracy of the detection result, a quality control sample is arranged in each fluorescent quantitative PCR reaction. The quality control product A is an individual sample of an iCre transgenic mouse with the copy number of the iCre gene of 1 through whole genome sequencing, the quality control product B is prepared by connecting a 336bp iCre gene fragment and a 191bp IL-2 fragment into a pUCm-T vector in front and back through a DNA sequence chemical synthesis method, and the plasmid vector is synthesized by Shanghai, and the structure schematic diagram is shown in figure 6. Namely, the quality control product A is a sample with a CNR value of 0.5, and the quality control product B is a sample with a CNR value of 1. And detecting the quality control product and the unknown sample together during each fluorescent quantitative PCR reaction, and calculating the CNR value of the quality control product and the unknown sample. When the CNR value calculated on the quality control product is within +/-5% of the true value, the detection result is effective, and the current detection meets the requirements.

2. Application instance

And detecting the unknown sample by using the standard product successfully constructed. The assay samples were CYP19A1-iCre transgenic mice constructed in the laboratory. The mice are transgenic mice which are over-expressed by randomly inserting CYP19A1-iCre gene sequences into mouse genome by using a PiggyBAC transposase system and adopting a fertilized egg injection mode. After obtaining the F0-generation founder mice, they were passaged mating with wild-type mice, which were now transferred to generation 6.

DNA is extracted by cutting rat tail tissue, the fluorescence quantitative PCR detection system established in the prior art is adopted for detection, and Ct is read _{Target gene} And Ct _{Control genes} The calculation was performed according to the above-mentioned formulas (I), (II) and (III).

First, R of a standard curve obtained by each fluorescent quantitative PCR reaction is ensured ² The value and the CNR value of the quality control product meet the requirements. The results are shown in Table 1, and the standard curve R of 10 experiments is counted ² The value can reach more than 0.99. As shown in table 2, the CNR values of quality control products of 10 experiments were counted, the CNR value of quality control product a was floated up and down around 0.5, and the CNR value of quality control product B was floated up and down around 1, which were all satisfactory.

Next, F0-generation Foundar mice were examined. The results in table 3 show the copy number of the cre transgene in 18F 0 mice. All F0 mice were identified as having multiple copies of the cre transgene. Of these, mouse No.022 contained the lowest 7.506 copies of the cre transgene, while mouse No.014 had the highest copy number of the cre transgene, 14.866 copies.

After subculturing these F0 mice, a total of 3F 0 mice could be stably passaged and the copy number of the cre gene in their individuals gradually decreased to a single copy. As shown in table 4, the copy number of the cre transgene gradually decreased with increasing passage number, and tended to stabilize after becoming a single copy. Wherein, for the F0 generation No.017 mice, the iCre transgene contained therein was no longer altered after passage to 2 copies. This is probably because the two copies of the cre gene are located exactly at the site of linkage inheritance.

TABLE 1 Standard curve R ² Statistics of values

TABLE 2 statistics of quality control CNR values

TABLE 3 copy number of the iCre transgene in the genome of individual F0 mice

TABLE 4.3 copy number of the iCre transgene in F0 mice and offspring individuals thereof

The standard product for detecting the copy number of the iCre transgene in the genome constructed by the invention is suitable for all model animals (such as mice, rats, rabbits, dogs and the like) which adopt a random transgene method to express the iCre gene. The established standard can be used for rapidly and accurately detecting and quantifying the copy number of the cre gene in the genome of the individual, and is greatly helpful for the establishment and application of the cre transgenic mode animal.

Claims

1. The preparation method of the universal standard for determining genome iCre transgene copy number based on absolute quantitative PCR is characterized by comprising the following steps:

2) The copy number of the iCre gene obtained by the first round of PCR amplification is n ₁ The fragment of (2) is connected into pUCm-T vector to obtain recombinant plasmid 1, the recombinant plasmid cloned with 2 XiCre fragment or 4 XiCre fragment is used as DNA template for second amplification of megaprimerPCR reaction to make second amplification, and the copy number of iCre gene is separated from the obtained PCR product ₂ Wherein said n ₂ Including any one largeAn even number of 4 or less and 32 or less; n obtained by separation ₂ The x iCre tandem repeat fragment is also ligated into pUCm-T vector to obtain recombinant plasmid 2;

2. The method of manufacturing according to claim 1, characterized in that: said n ₂ Is an integer multiple of 4, preferably n ₂ =4, 8,16 or n ₂ =4, 8,16,32 or n ₂ =8, 16 or n ₂ = 8,16,32; preferably n ₁ ＝1,2,4，n ₂ ＝8,16。

3. The method of manufacturing according to claim 1, characterized in that: the primers used for PCR amplification in steps 1) and 2) are MegP-F:5' -TGAACGCCACTGACTTTGAC-3, megP-R:5'-CTTGTCAAAGTCAGTGCGTTCAGCAGCCACACCATTCTTTCT-3';

4. The method of manufacturing according to claim 1, characterized in that:

adopting a PCR product direct TA connection method in the step 2), and setting the copy number of the iCre gene as n ₁ 、n ₂ Adding A at two ends of the PCR product, and connecting the product obtained by the reaction of adding A into a pUCm-T vector through TA cloning;

5. The method of manufacturing according to claim 1, characterized in that: in the step 3), the sequences of the amplification primer pairs of the IL-2 gene fragment 1 are shown as SEQ ID NO.5 and SEQ ID NO.6, and the sequences of the amplification primer pairs of the IL-2 gene fragment 2 are shown as SEQ ID NO.7 and SEQ ID NO. 8.

6. A universal standard prepared by the method of any one of claims 1 to 5.

7. A method for determining the copy number of an cre transgene based on an absolute quantitative PCR method, wherein the determination is performed by using the universal standard according to claim 6, comprising the steps of:

1) Establishing a standard curve:

ΔCt＝Ct _{target gene} -Ct _{Control genes} The (I)

y ₁ ＝ax ₁ +b, formula (II)

In the formula (II), x ₁ Is log of ₂ (CNR) value, y ₁ Delta Ct value;

2) Detecting a sample to be detected:

y ₂ ＝x ₂ *2 ^x1 the (III)

In the formula (III), y ₂ For the actual copy number, x, of the cre gene in the genome of the sample organism to be tested ₂ Is the chromosome multiple of the organism to which the sample to be measured belongs.

8. The method according to claim 7, wherein: and when a sample to be detected is detected, a quality control sample is used, wherein the quality control sample comprises a quality control product A and/or a quality control product B, the quality control product A is an individual sample of an iCre transgenic mouse with the copy number of the iCre gene being 1 through whole genome sequencing, and the quality control product B is a plasmid sample with the CNR of the iCre/IL-2 being 1.

9. The method according to claim 7, wherein: the species to which the sample to be tested belongs is a mouse, the fluorescent quantitative PCR amplification primer sequences of the iCre gene are shown as SEQ ID NO.9 and SEQ ID NO.10, and the fluorescent quantitative PCR amplification primer sequences of the IL-2 gene are shown as SEQ ID NO.11 and SEQ ID NO. 12;

the reaction system is as follows:2 XQuantiNovaSYBRGreenPCRMastermix 10. Mu.l each, forward/reverse primer 0.5. Mu.l each, DNA template 1. Mu.l, ddH ₂ O was made up to 20. Mu.l;

10. The method according to any one of claims 1 to 9, wherein: the ORF sequence of the iCre gene is shown as SEQ ID NO.1, and the IL-2 gene amplification sequence is shown as SEQ ID NO. 4;