CN118086394B - Pig adipose tissue development tracing system and application - Google Patents
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Abstract
The invention relates to the technical field of biology, and provides a pig adipose tissue development tracing system and application thereof, wherein the system comprises two vectors of px330-Rosa26 and pRosa-AdipoTracer-KI. Cells or individuals of AdipoTracer systems are integrated in the pig friendly locus Rosa26 at fixed points, and the constructed pig fat tracer system can respond to 4-hydroxy tamoxifen (4-OHT) induction, so that the development and development processes of pig fat cells are traced, the pig fat tracer system has important significance for breeding pigs and other large-scale livestock, and has important reference value for treating human obesity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a pig adipose tissue development tracing system and application thereof.
Background
The fat has very important biological significance, can provide fatty acid and energy for living bodies, not only plays a role in energy storage, but also plays a very important role in maintenance and metabolic regulation of energy steady state. Meanwhile, fat is an indispensable substance for forming new tissues and repairing old tissues in the organism, is an important endocrine organ, and has physiological functions of protecting viscera, preserving heat of the organism and the like.
In the live pig industry, fat deposition of pigs is one of the complex economic traits, and is closely related to backfat thickness, lean meat percentage and feed conversion efficiency, and meat quality is influenced by intramuscular fat content. The genetic nature understanding of the development characteristics of pig fat and the analysis of the regulation and control mechanism of fat specific deposition at different parts are key to realizing the cooperative breeding of lean meat percentage and meat quality, and are the premise and basis for cultivating low-fat high-quality pig varieties. At present, the control mechanism of directional deposition of adipose tissues of pigs is not clear, and there is an urgent need to develop a genetic lineage tracing technology (GENETIC LINEAGE TRACING) of adipose tissues for revealing the fat deposition process of pigs.
Genetic lineage tracing technology is a method of using a specific method to label a specific cell or a specific class of cells, and the system tracks and displays the cell fate, and is used to reveal the phenomenon of self-renewal, differentiation and transdifferentiation of specific cell types in development, disease and regeneration. Currently, this technology has been widely used in model animals. Compared with mice, the construction of a genetic tracing pig system faces two difficulties, firstly, two to three transgenic mice carrying different elements are required to be bred for constructing the genetic tracing mice, and the period of constructing the pig tracing system according to the strategy is very long and is difficult to realize; secondly, the effective porcine adipocyte specific promoter has not been verified, which is easy to cause interference of non-target cell signals, has low signal-to-noise ratio and poor resolution.
Therefore, the development of a novel pig fat tracer system analyzes the fat deposition process of pigs, has important significance for pig breeding and has important reference value for treatment of human obesity.
Disclosure of Invention
The invention aims to provide a pig adipose tissue development tracing system and application thereof.
In order to achieve the aim of the invention, in a first aspect, the invention provides a pig adipose tissue development tracing system, which consists of a pig adipose tracing carrier and a targeting carrier;
The pig fat tracer vector comprises a sequence consistent with the sgRNA of a targeted pig Rosa26 gene (namely pRosa protospacer sequence, the reverse complementary sequence of which can be specifically identified by the sgRNA sequence), a CRE ERT2 element for pAdipoq expression initiation and a mTmG element for pCAG expression initiation, and the nucleotide sequences of the elements are formed by sequentially connecting SEQ ID NO. 6-8; the targeting vector is a targeting vector which comprises an sgRNA sequence of a targeted pig Rosa26 gene and expresses Cas9 protein, and the nucleotide sequence of the targeting vector is shown as SEQ ID NO. 9.
The porcine adipose tissue development tracer system of the invention can respond to 4-hydroxy tamoxifen (4-OHT) induction.
In a second aspect, the invention provides the use of the system in the tracing of porcine adipose tissue developmental lineages.
In a third aspect, the present invention provides a method of tracing a porcine adipose tissue developmental lineage, the method comprising: and co-transfecting the pig fetal fibroblasts with the pig adipose tracer vector and the targeting vector, obtaining a monoclonal cell line with a pig adipose tissue development tracer system integrated at fixed points through flow cell sorting and genotyping, and preparing a transgenic pig through a somatic cell nuclear transfer technology so as to trace the development process of adipose tissues.
Further, primers used for genotyping were as follows:
5-F: 5’-CCTCCGGGGGAGTCGGTTTA-3’，
5-R is 5'-TTTCCTCCTCTCCTGACTAC-3'; and
3-F: 5’-CCGCCGCGCTTAATGCGCCG-3’，
3-R: 5’-AGGTCCAATCGCAGTGGTAG-3’。
The invention provides a pig fat tracer system (namely a pig adipose tissue development tracer system), which is used for integrating the pig fat tracer system at fixed points in a Rosa26 friendly gene locus of an isolated fibroblast fetal cell genome of a target animal, and can respond to 4-OHT induction and permanently mark mature adipocytes as green in a adipogenic differentiation process so as to trace the occurrence and development processes of the pig fat cells.
The site-directed integration can cleave the porcine Rosa 26-friendly gene site by CRISPR/Cas9, TALEN, or ZFN. The fat specific expression pRosa-AdipoTracer-KI vector (namely the pig fat tracer vector) is integrated at fixed points in the pig Rosa26 friendly gene locus by combining with a non-homologous recombination dependent fixed point integration strategy.
Further, the identification and cleavage of the pig Rosa26 friendly gene locus is carried out by CRISPR/Cas9, the CRISPR/Cas9 vector px330-Rosa26 shown in SEQ ID NO. 9 has the nucleotide sequence GTGAGAGTTATCTGACCGTA from 250 th to 269 th of the 5' end as sgRNA-pRosa spacer, the corresponding specific identification region has the nucleotide sequence SEQ ID NO. 5 from 1640 th to 1659 th of the 5' end as well as the nucleotide sequence SEQ ID NO. 7 from 7128 th to 7147 th of the 5' end.
Further, the site-directed integration vector pRosa-AdipoTracer-KI vector sequence is formed by sequentially connecting SEQ ID NO: 6-8, wherein the combination of regulatory elements is innovations of the invention, nucleotide sequences 4-66 from the 5' end of SEQ ID NO:6 are PiggyBac 3' inverted repeat sequences, nucleotide sequences 663-2264 from the 5' end of SEQ ID NO:6 are pCAG sequences, nucleotide sequences 2366-5678 from the 5' end of SEQ ID NO:6 are mTmG (LoxP-mT-LoxP-mG) sequences for expressing red fluorescent protein, nucleotide sequences 6794-10000 from the 5' end of SEQ ID NO:6 and nucleotide sequences 1-2284 from the 5' end of SEQ ID NO:7 are fat specific promoter pAdipoq sequences, nucleotide sequences 2308-5229 from the 5' end of SEQ ID NO:7 are Cre ERT2 sequences, nucleotide sequences 7050-7084 from the 5' end of SEQ ID NO:7 are sequence of PiggyBac 35, and nucleotide sequences 7124 from the 5' end of SEQ ID NO:7 are sequence of PiggyBac 3' inverted repeats from the 5' end of SEQ ID NO: 7.
Further, the primers in table 1 are adopted to carry out PCR amplification on the monoclonal cells, PCR amplification products are detected, and monoclonal cells with the fragment sizes of the amplification products being the corresponding values of the fixed-point integration product sizes are selected to carry out sequencing, and the sequencing result is AdipoTracer system fixed-point integration cells with correct sequencing results.
TABLE 1 identification primer information Table
Further, fetal fibroblasts prepared according to the method and containing AdipoTracer system site-directed integration were obtained by somatic cell nuclear transfer to fat tagged pigs.
The animal of the invention is a mammal, in particular a pig, and the tissue is in particular fat.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
The invention provides construction and application of a pig fat tracer system, which comprises two vectors, namely px330-Rosa26 and pRosa-AdipoTracer-KI. Cells or individuals of AdipoTracer systems are integrated in the pig friendly locus Rosa26 at fixed points, and the constructed pig fat tracer system can respond to 4-OHT induction, so that the development and development processes of pig fat cells are traced, the pig fat tracer system has important significance for breeding pigs and other large-scale domestic animals, and has important reference value for treating human obesity.
Drawings
FIG. 1 is a schematic diagram showing the construction and cell verification of a fluorescent tracer (AdipoTracer) system specific for mature fat in response to 4-OHT induction in accordance with a preferred embodiment of the invention; wherein A is a schematic diagram of a mature fat specific tracer system PB-AdipoTracer; b sorting tdTomato positive 3T3-L1 preadipocyte line for FACS; C4-OHT treatment differentiated 3T3-L1 preadipocytes to produce mature adipocytes that express EGFP.
FIG. 2 is a schematic representation of the design and selection of high efficiency sgRNAs targeting porcine Rosa26 sites in accordance with a preferred embodiment of the present invention.
FIG. 3 is a diagram showing the selection and identification of AdipoTracer site-directed integration of PEF monoclonal cell lines in accordance with a preferred embodiment of the present invention; wherein A is the construction flow of a AdipoTracer site-directed integrated PEF monoclonal cell line; b is the gel electrophoresis identification result of AdipoTracer-KI-1# positive monoclonal cell line PCR products; c is the Sanger sequencing result of AdipoTracer-KI-1# positive monoclonal cell line at the integration site.
FIG. 4 is a schematic illustration of the AdipoTracer system of the preferred embodiment of the present invention in the adipogenic differentiation of PEF cells; wherein A is the lipid-forming differentiation schematic diagram of a Rosa26-AdipoTracer-KI-PEF monoclonal cell line; b is the results of oil red O and fluorescence photographs of Rosa26-AdipoTracer-KI-PEF monoclonal cell line when treated with WAT inducer and 4-OHT for 15: 15 d.
Detailed Description
The invention provides a pig fat tracer system: first, a mature fat specific tracer system was constructed, comprising a Piggybac (PB) transposon plasmid (PB-AdipoTracer) containing a CRE ERT2 element whose expression is initiated by a fat specific promoter pAdipoq and a mTmG element whose expression is initiated by pCAG, and a PB transposase plasmid (Super PB), both plasmids were co-transfected into a mouse preadipocyte line 3T3-L1 for verification, and the system was found to normally respond to induction by 4-hydroxy tamoxifen (4-OHT), indicating that the system was successfully constructed.
Secondly, aiming at a first intron sequence of a friendly gene locus Rosa26 of pigs, designing effective sgRNA, and constructing a corresponding clustered regular interval short palindromic repeat sequence and a related protein 9（clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9, CRISPR/Cas9） carrier px330-Rosa26 thereof; construction of a pRosa-AdipoTracer-KI vector containing the pRosa protospacer sequence, the CRE ERT2 element for expression of fat-specific promoter pAdipoq and the mTmG element for pCAG; co-transfecting pig fetal fibroblasts (porcine embryo fibroblast, PEF) with a px330-Rosa26 vector and a pRosa26-AdipoTracer-KI vector, screening a fixed-point integrated Rosa26-AdipoTracer-KI-PEF cell line by a CRISPR/Cas9 mediated non-homologous recombination dependent fixed-point Integration (HITI) strategy, wherein the Cas9 on the px330-Rosa26 vector can not only recognize and cut a first intron sequence in a Rosa26 friendly gene locus on a PEF cell genome, but also specifically recognize and cut a pRosa protospacer sequence on a pRosa26-AdipoTracer-KI vector, thereby achieving the aim of linearizing the pRosa26-AdipoTracer-KI vector; we finally successfully screened the Rosa26-AdipoTracer-KI-PEF monoclonal cell line by flow cell sorting (Fluorescence ACTIVATING CELL Sorter, FACS) and genotyping.
Finally, adipogenic differentiation and 4-OHT treatment were performed on the Rosa26-AdipoTracer-KI-PEF monoclonal cell line obtained by screening, further verifying that AdipoTracer system was able to respond effectively to 4-OHT induction in porcine cells. Based on the above, the invention focuses on the construction of a pig fat specific tracer system and the application of the pig fat specific tracer system in analysis of development rules, cell compositions and molecular regulation mechanisms of pig fat tissues.
The invention provides an application of a pig fat tracer system in at least one of the following 1) -5):
1) Tracing in the adipogenic differentiation process of mouse preadipocytes;
2) Cleavage of the Rosa 26-friendly gene locus in the pig genome;
3) Tracing in the process of adipogenic differentiation of porcine preadipocytes;
4) Tracing during adipogenic differentiation of porcine PEF cells;
5) Preparation of fat-specific tracer gene editing pigs.
Preferably, the PB-AdipoTracer transposable vector sequence containing pAdipoq CRE ERT2 element for promoting expression and mTmG element for promoting pCAG expression is formed by sequentially connecting SEQ ID NO. 1-3.
Preferably, the PB transposase plasmid Super PB vector sequence is shown in SEQ ID NO. 4.
Preferably, the nucleotide sequence specifically recognized by sgRNA in the first intron of the Rosa26 gene locus in the pig genome is shown as SEQ ID NO. 5.
Preferably, the pRosa-AdipoTracer-KI vector containing pRosa protospacer sequence, CRE ERT2 element for pAdipoq expression promotion and mTmG element for pCAG expression promotion is formed by sequentially connecting SEQ ID NO. 6-8.
Preferably, the px330-Rosa26 vector sequence which specifically recognizes and cleaves the first intron sequence of the Rosa26 friendly gene locus in the pig genome, the pRosa protospace sequence in pRosa-AdipoTracer-KI vector is shown as SEQ ID NO. 9.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
Example 1 construction and cell verification of a fluorescent tracer (AdipoTracer) System specific for mature fat in response to 4-OHT induction
PB transposon plasmid PB-AdipoTracer (FIG. 1A, the sequences of which are sequentially connected by SEQ ID NO: 1-3) containing CRE ERT2 element whose expression is promoted by fat specific promoter pAdipoq and mTmG element whose expression is promoted by pCAG was constructed, and co-transfected with PB transposase plasmid Super PB (SEQ ID NO: 4) into 3T3-L1 mouse preadipocyte line. After long-term culture for 16d, cells accounting for 16.9% of the total cell integration pAdipoq-Cre ERT2 -pCAG-mTmG system, tdTomato red fluorescent protein was continuously expressed (fig. 1B). Red fluorescent-labeled 3T3-L1 preadipocytes were sorted, enriched, and simultaneously subjected to white fat (WAT) cell differentiation and 4-OHT treatment by FACS: treatment was performed with 3T3-L1 white adipocyte differentiation medium (DMEM/F12 basal medium containing 10% FBS, 1% Streptomyces lividans (PS), 20mM HEPES, 5 μg/mL insulin, 1 μM dexamethasone, 0.5mM isobutylmethylxanthine and 1 μM rosiglitazone) for 4 days. Subsequently, 3T3-L1 white adipocyte maturation medium (DMEM/F12 basal medium containing 10% FBS, 1% PS, 5 μg/mL insulin, and 1 μg/mL dexamethasone) was used for 4 days, with fresh maturation medium replaced every 2 days. 4-OHT induced group cells, wherein 4-OHT drug treatment is continuously carried out in the process of adipogenic induced differentiation, namely 1 [ mu ] M4-OHT is additionally added into white adipocyte differentiation and maturation medium. It was verified by fluorescence detection analysis whether AdipoTracer system was able to respond efficiently to 4-OHT induction in mouse cells, giving rise to a tdTomato red fluorescence to EGFP green fluorescence conversion.
The mature adipocytes can be permanently marked as green by 4-OHT induction, and the mature adipocytes which are newly grown after the induction is stopped are permanently marked as red, so that the amount of red fluorescent markers can reflect the proliferation degree of the cells, and the size change of the adipocytes marked by green fluorescent markers can reflect the hypertrophy change of the cells. Therefore, the key time law and characteristic difference of adipose tissue development can be revealed.
Fluorescence photography was performed at induction number 8 d, and it was found that induction of differentiated mature adipocyte expression enhanced green fluorescent protein (ENHANCED GREEN Fluorescent Protein, EGFP) (fig. 1C). The above results indicate that a fluorescent tracer plasmid vector specific for mature fat in response to 4-OHT induction has been successfully constructed.
Example 2 design and screening of efficient sgrnas targeting the porcine Rosa26 site
Targeting sgrnas were designed for the first intron sequence in the Rosa26 friendly locus on the pig genome using the sgRNA design and evaluation website CRISPOR (http:// crispor. Tefor. Net /), from which 3 websites were selected for validation of sgrnas (numbered sgrnas 1-3) with high editing efficiency and low off-target activity in PEF cells of pigs (fig. 2A). And respectively constructing px330-sgRNA 1-3 vectors, transfecting the vectors into PEF cells of pigs, collecting mixed cells at 48h of transfection, extracting cell DNA, and amplifying target fragments by PCR, wherein the primers are 5-F (SEQ ID NO: 10) and 3-R (SEQ ID NO: 13). Mixed cell editing efficiency was tested by Sanger sequencing and editing efficiency analysis website TIDE (https:// TIDE. Nki. Nl /). Editing efficiency analysis results found that the efficiency of px330-sgRNA3 cleavage of the pig Rosa26 friendly locus was significantly higher than that of px330-sgRNA1 and px330-sgRNA2 (fig. 2B). Thus, it is preferred that px330-sgRNA3 specifically recognizes and site-specifically cleaves the first intron sequence in the Rosa26 friendly gene locus on the pig genome, designated px330-Rosa26 vector (SEQ ID NO: 9).
Example 3 construction of AdipoTracer System site-directed integration into porcine Rosa26 site and screening and identification of PEF monoclonal cell lines
PRosa26 protospacer (GTGAGAGTTATCTGACCGTAAGG, the underlined region is NGG sequence) is designed aiming at the first intron region (SEQ ID NO: 5) of the Rosa26 friendly locus in pig gene pigs, and the sequence is constructed to a donor vector to finally form pRosa-AdipoTracer-KI (which is formed by sequentially connecting SEQ ID NO: 6-8). The vector also contains the CRE ERT2 element for pAdipoq to promote expression and the mTmG element for pCAG to promote expression.
As pRosa-AdipoTracer-KI vector also contains pRosa protospacer sequence, the sequence can be identified and cut by the specificity of px330-Rosa26 vector, thereby achieving the purpose of linearizing pRosa-AdipoTracer-KI vector.
Two vectors pRosa-AdipoTracer-KI and px330-Rosa26 were co-transfected into porcine PEF cells. Cell culture was performed by CRISPR/Cas9 mediated HITI non-homology dependent site-directed integration strategy using FACS sorting of monoclonal cells (fig. 3A). After 10d of culture, genotyping the well-grown monoclonal cell line, including site-directed integration of the 5 'junction sequence, the 3' junction sequence, and the site-directed integration of the full-length sequence, and screening AdipoTracer for positive PEF monoclonal cell lines site-directed integration at the Rosa26 site. Genotyping primer information is shown in Table 1.
The amplification and clone sequencing results of the PCR products show that the fixed-point integration of one chromosome (Allle 1) of the 1 # cell line is realized, the size of the amplified product at the 5 'joint is 1739bp, 1bp insertion exists, the size of the amplified product at the 3' joint is 656bp, and 6bp deletion exists (figures 3B and 3C); the other chromosome (Allele 2) was not site-specific but had a 1bp deletion in the target site. Meanwhile, the length of the pre-amplified fragment of the full-length identification primer integrated at fixed point reaches 23197bp, so that the pre-amplified fragment is difficult to amplify, and only a 1301bp fragment-sized band is amplified. The above results indicate that the 1 # cell line is a AdipoTracer-KI heterozygous knock-in cell line, designated as the Rosa26-AdipoTracer-KI-PEF monoclonal cell line.
Example 4 AdipoTracer verification of System in the adipogenic differentiation of PEF cells
The Rosa26-AdipoTracer-KI-PEF monoclonal cell line expressed red fluorescence when untreated. It was subjected to simultaneous WAT inducer and 4-OHT treatment: 1 [ mu ] M4-OHT drug was added to white adipocyte differentiation medium (DMEM basal medium containing 10% FBS, 1% PS, 20mM HEPES, 5 [ mu ] g/mL insulin, 17 [ mu ] M pantothenic acid, 33 [ mu ] M biotin, 1 [ mu ] M dexamethasone, 0.25mM isobutyl methylxanthine and 1 [ mu ] M rosiglitazone) and treated for 5 days. Subsequently, 1 [ mu ] M4-OHT drug was added to white adipocyte maturation medium (DMEM basal medium containing 10% FBS, 1% PS, 5 [ mu ] g/mL insulin, 17 [ mu ] M pantothenic acid, 33 [ mu ] M biotin and 1 [ mu ] M dexamethasone) for 10 days, with fresh maturation medium replaced every 3 days. Oil red O staining and fluorescence photography were performed at induction 15d (fig. 4A). As a result, it was found that this cell line was able to differentiate into a large amount of oil red O-stained mature adipocytes at the time of adipogenic induction for 15d (FIG. 4B). In the adipogenic differentiation process, the mature adipocytes generated by adding 4-OHT treatment can be converted from tdTomato red fluorescence to GFP green fluorescence; whereas mature adipocytes not subjected to 4-OHT treatment expressed only red fluorescence (fig. 4B).
The invention successfully induces and differentiates PEF cells into mature adipocytes, and further verifies that AdipoTracer systems can effectively respond to 4-OHT induction in pig cells, thereby laying a foundation for preparing AdipoTracer fixed-point integrated pigs by subsequent combined body nuclear transfer (SCNT) and embryo transfer technology.
Sequence description:
1. The PB-Adipotracer (PB-pCAG-mTmG-pAdipoq-Cre ERT2) vector sequence is formed by sequentially connecting SEQ ID NO 1-3, wherein the nucleotide sequence of SEQ ID NO 1 from the 5' -end at positions 4-66 is a PiggyBac 3' inverted repeat sequence, the nucleotide sequence of SEQ ID NO 1 from the 5' -end at positions 663-2264 is a pCAG sequence, the nucleotide sequence of SEQ ID NO 1 from the 5' -end at positions 2366-5678 is a mTmG (LoxP-mT-LoxP-mG) sequence for expressing red fluorescent protein, the nucleotide sequence of SEQ ID NO 1 from the 5' -end at positions 6794-10000 and the nucleotide sequence of SEQ ID NO 2 from the 5' -end at positions 1-2284 is a fat specific promoter pAdipoq sequence, the nucleotide sequence of SEQ ID NO 2 from the 5' -end at positions 2308-5229 is a Cre ERT2 sequence, and the nucleotide sequence of SEQ ID NO 2 from the 5' -end at positions 7050-7084 is a PiggyBac 5' inverted repeat sequence.
2. Super PB (pCAG-PBase) vector sequence SEQ ID NO. 4, wherein the base sequence of 4-1670 is pCAG sequence, and the nucleotide sequence of 1735-3519 is PBase sequence.
3. In the first intron of the Rosa26 gene locus in the pig genome, the nucleotide sequence specifically recognized by sgRNA is SEQ ID NO. 5, wherein nucleotide sequences 1-124 are exon 1 regions, nucleotide sequences 125-2000 are partial intron 1 sequences, and nucleotide sequences 1640-1659 are regions specifically recognized by sgRNA.
4. PRosa-AdipoTracer-KI (PB-pCAG-mTmG-pAdipoq-Cre ERT2- pRosa-protospacer) vector sequence is formed by connecting SEQ ID NO 6-8 in sequence, wherein the nucleotide sequence of SEQ ID NO 6 from the 5 'end at positions 4-66 is a PiggyBac 3' inverted repeat sequence, the nucleotide sequence of SEQ ID NO 6 from the 5 'end at positions 663-2264 is a pCAG sequence, the nucleotide sequence of SEQ ID NO 6 from the 5' end at positions 2366-5678 is a mTmG (LoxP-mT-LoxP-mG) sequence for expressing red fluorescent protein, the nucleotide sequence of SEQ ID NO 6 from the 5 'end at positions 6794-10000 and the nucleotide sequence of SEQ ID NO 7 from the 5' end at positions 1-2284 is a fat specific promoter pAdipoq sequence, the nucleotide sequence of SEQ ID NO 7 from the 5 'end at positions 8-5229 is a Cre 2 sequence, the nucleotide sequence of SEQ ID NO 7 from the 5' end at positions 7050-7084 is a nucleotide sequence of SEQ ID NO 7 from the 5 'end at positions 7050-7084, and the nucleotide sequence of SEQ ID NO 7 from the 5' end at positions 7142 is a sequence of PiggyBac 2.
5. The vector sequence of px330-pRosa is SEQ ID NO. 9, wherein the nucleotide sequence at 250-269 is the sgRNA-pRosa spacer sequence.
6. The 5-F/5-R primer sequence is shown in SEQ ID NO. 10-11, and the 3-F/3-R primer sequence is shown in SEQ ID NO. 12-13.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (5)
1. The pig adipose tissue development tracing system is characterized by comprising a pig adipose tracing carrier and a targeting carrier;
The pig fat tracer vector comprises a sequence consistent with sgRNA of a targeted pig Rosa26 gene, a CRE ERT2 element for pAdipoq to start expression and a mTmG element for pCAG to start expression, and the nucleotide sequences of the pig fat tracer vector are formed by sequentially connecting SEQ ID NO. 6-8;
The targeting vector is a targeting vector which comprises an sgRNA sequence of a targeted pig Rosa26 gene and expresses Cas9 protein, and the nucleotide sequence of the targeting vector is shown as SEQ ID NO. 9.
2. The system of claim 1, wherein the system is responsive to induction by 4-hydroxy tamoxifen.
3. Use of the system of claim 1 or 2 for tracing porcine adipose tissue developmental lineages.
4. A method of tracing a porcine adipose tissue developmental lineage, the method comprising: co-transfecting the pig fetal fibroblasts with the pig adipose tracer vector and the targeting vector according to claim 1 or 2, obtaining a monoclonal cell line with a pig adipose tissue development tracer system integrated at fixed points through flow cell sorting and genotyping, and preparing a transgenic pig through a somatic cell nuclear transfer technology to trace the development progress of adipose tissue.
5. The method according to claim 4, wherein the primers used for genotyping are as follows:
5-F: 5’-CCTCCGGGGGAGTCGGTTTA-3’，
5-R is 5'-TTTCCTCCTCTCCTGACTAC-3'; and
3-F: 5’-CCGCCGCGCTTAATGCGCCG-3’，
3-R: 5’-AGGTCCAATCGCAGTGGTAG-3’。
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