Application of exogenous gene integration site, homologous recombinant vector and construction method of transgenic pig

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of an exogenous gene integration site, a homologous recombinant vector and a construction method of a transgenic pig. According to the present invention, the safety site integrated by the exogenous gene provided by the present invention is the region between the LOC100155859 tubulin alpha-1B chain-like gene and the LOC100517052 histone H3.3-like gene of the pig chromosome 11, and is the region between the LOC100155859 tubulin alpha-1B chain-like gene and the LOC100517052 histone H3.3-like gene of the pig chromosome 11; the safe site is further fully verified on transgenic animals and cells, and the exogenous gene can be stably and efficiently expressed in the passage process and has no side effect on the cells and tissues. Therefore, the safe site provided by the invention has a good application prospect in gene engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a use of an exogenous gene integration site, a homologous recombination vector and a method for constructing a transgenic pig. Background Art

[0002] At present, the most commonly used method for constructing stable cell lines and transgenic animals is to integrate the target gene into a safe site of the genome or randomly integrate it, and then obtain the recombinant cell line that meets the requirements through a large number of screening. One of the difficulties of this method is the variability and uncertainty of the random integration site of the target gene, which may lead to the loss of the target gene copy number and gene rearrangement in the recombinant cell line during long-term culture, thereby affecting the expression stability of the cell in the later stage.

[0003] Here, the concept of site effect is proposed. Site effect refers to the direct correlation between the integration site of the exogenous target gene and the characteristics of the cell line (such as the expression level of the exogenous target gene and the stability of the recombinant cell). Related studies have pointed out that site effect can have enhancing and weakening effects on the expression of exogenous target genes. Different gene integration sites have different structures and regulatory backgrounds, and the active gene expression area is generally in the open area of ​​the chromosome. Therefore, the target gene is affected by different integration sites, which will make the expression level and stability of the recombinant cell line in long-term culture different.

[0004] In order to ensure the safety of exogenous gene insertion and normal expression, the development of safe sites (Genomic Safe Harbors, GSHs) on animal genomes is an important content. Safe sites provide a safe and reliable "port (Safe Harbor)" for exogenous genes, so that exogenous genes can be expressed stably and efficiently, and have no side effects on cell growth, embryonic development and individual health. Existing studies have found a number of safe sites that can be used for exogenous gene insertion in mammals (for example: AAVS1, CCR5, HPRT, H11, Col1a1, TIGRE, etc.), and these safe sites have been applied to humans, mice, rats, and rabbits to achieve regulatable or reversible gene overexpression. Among them, H11 and AAVS1 sites are also used as safe sites in the pig genome.

[0005] However, the expression level of exogenous genes is closely related to the insertion site. Studies have found that the insertion sites within certain genes cannot achieve high-level expression of exogenous genes; some gene sites (such as GGTA1 and CMAH) cannot be effectively expressed after inserting exogenous genes; some so-called "safe sites" will show changes in gene expression during cell passage and lack stability. For example, studies have found that the insertion of reporter genes at the AAVS1 safe site has large expression and silencing variations among different human induced pluripotent stem cell (hiPSC) clones, which seriously affects the stability of gene expression. In addition, the area of ​​some safe sites is relatively small, making it difficult to insert large fragment vectors. For example, the H11 site is located between the two genes Eif4enif1 and Drg1, and its available interval is only 5 kb. The insertion efficiency of large fragments is extremely low, and it is still uncertain whether it can meet the integration requirements of larger fragments. In addition, the safety site is close to the upstream and downstream genes, which may have a potential impact on the expression of adjacent genes.

[0006] Therefore, developing new safe sites for exogenous gene integration is an important research topic in this field. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a use of an exogenous gene integration site, a homologous recombination vector and a method for constructing a transgenic pig.

[0008] The use of the safe site gene fragment located at 30963218-32221030 of pig chromosome 11 in the site-specific integration of foreign genes in the pig genome, the genome data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5.

[0009] Preferably, the safety site gene fragment is located at 31250963-31252424 of pig chromosome 11.

[0010] Preferably, the safety site gene fragment is the region between tubulin alpha-1B chain-like and histoneH3.3-like genes.

[0011] Preferably, in the safety site gene fragment, the nucleotide sequence of the site for inserting the foreign gene is as shown in any one of SEQ ID NO.1 to SEQ ID NO.6.

[0012] The present invention also provides a homologous recombination vector for site-specific integration of foreign genes in a safe site gene fragment, wherein the safe site gene fragment is located at 30963218-32221030 of pig chromosome 11, and the genome data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5; The homologous recombination vector comprises a promoter sequence, a foreign gene fragment and a transcription termination signal sequence.

[0013] Preferably, the promoter sequence is shown as SEQ ID NO.7 or SEQ ID NO.12; and / or, the transcription termination signal sequence is shown as SEQ ID NO.9 or SEQ ID NO.14.

[0014] Preferably, the homologous recombination vector further comprises a left homology arm and a right homology arm.

[0015] Preferably, the nucleotide sequence of the left homology arm is shown as SEQ ID NO.10, and the nucleotide sequence of the right homology arm is shown as SEQ ID NO.11; Alternatively, the nucleotide sequence of the left homology arm is as shown in SEQ ID NO.15, and the nucleotide sequence of the right homology arm is as shown in SEQ ID NO.16.

[0016] The present invention also provides a method for constructing a transgenic pig, which integrates a foreign gene in a safe site gene fragment, wherein the safe site gene fragment is located at 30963218-32221030 of pig chromosome 11, and the genome data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5.

[0017] Preferably, the method comprises the following steps: Cultivating donor cells; transferring a homologous recombination vector for integrating foreign genes into the donor cell; Identify positive clones; The positive cell clones obtained were used for somatic cell nuclear transplantation and embryo transplantation.

[0018] The present invention provides a safe site for the stable integration of foreign genes for the pig genome. It has been verified on transgenic animals that after the foreign genes are inserted into the safe site provided by the present invention, the foreign genes can be stably and efficiently expressed during the passage process, and have no side effects on cells and tissues. In addition, the safe site area reaches 2Mb, which supports the insertion of long fragments, and the safe site is very far away from the upstream and downstream genes, so the potential risk of affecting gene expression is low. Therefore, the safe site provided by the present invention has a good application prospect in genetic engineering.

[0019] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0020] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the location of the region between the tubulin alpha-1B chain-like gene LOC100155859 and histone H3.3-like gene LOC100517052 on chromosome 11 of pig; Figure 2 This is a diagram of the construction of the homologous recombination vector in Experimental Example 1; Figure 3 The results of the detection of exogenous gene expression in F0 generation cloned piglets in Experimental Example 1; Figure 4 The results of flow cytometry identification of transgenic expression in F0 cloned piglets in Experimental Example 1; Figure 5 The experimental results of detecting transgene expression in vascular endothelial cells of F0 cloned piglets in Experimental Example 1; Figure 6 The results of the detection of exogenous gene expression in F1 generation piglets in Experimental Example 1; Figure 7 The results of the detection of exogenous gene expression in F2 generation piglets in Experimental Example 1; Figure 8 The results of the detection of exogenous gene expression in F3 generation piglets in Experimental Example 1; Fig. 9 The experimental results of transgenic expression in F1-F3 generation piglets in Experimental Example 1; Fig.10 The results of transgenic expression in F1-F3 generation piglets in Experimental Example 1; Fig.11 Schematic diagram of the insertion position of the transgenic expression vector in Experimental Example 1; Fig.12 The diagram is a construction diagram of vector L and vector R in Experimental Example 1; Fig.13 These are the results of detecting the expression of exogenous genes in pig ear fibroblasts after vector L and vector R were transfected into the cells in Experimental Example 1. DETAILED DESCRIPTION

[0022] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0023] In the embodiment, the sequences involved are as follows: Table 1 Partial sequences involved in the examples Example 1 Safe site and homologous recombination vector for site-specific integration of foreign genes into the porcine genome This embodiment provides a safe site in the pig genome that can be used for site-specific integration of foreign genes, which is the region between the tubulin alpha-1B chain-like gene LOC100155859 and the histoneH3.3-like gene LOC100517052 on chromosome 11 of pig (Sus scrofa chromosome 11, 30963218-32221030), such as Figure 1 The blue frame part.

[0024] As a preferred solution, the safety site is located at Sus scrofa chromosome 11, 31250963-31252424.

[0025] The homologous recombination vector for inserting the foreign foreign gene in the safe site comprises a promoter sequence, a foreign gene fragment and a transcription termination signal sequence. As a preferred embodiment, the homologous recombination vector comprises a left homology arm, a promoter sequence, a foreign gene fragment, a transcription termination signal sequence and a right homology arm.

[0026] The method for constructing a transgenic pig based on the safety site comprises the following steps: A homologous recombination vector is constructed and used to transfect donor cells. After obtaining positive cell clones through DNA and RNA identification, the cells are used for somatic cell cloning. After obtaining cloned piglets, the DNA and protein expression of the piglets are identified, and high-expressing piglets are selected for breeding and subculture.

[0027] The technical solution of the present invention is further illustrated by experiments below.

[0028] Experimental Example 1: Verification of safe sites This experimental example verifies the safe sites provided in Example 1. The specific experimental process is as follows: 1. Determine the location of the safe site 1. Construction of exogenous gene insertion vector The porcine TM promoter sequence was amplified from the porcine genome by PCR, and its nucleotide sequence is shown as SEQ ID No. 7, which is used to drive the expression of exogenous genes.

[0029] 2. The human aortic endothelial cell line DNA was extracted by PCR, and the CDS sequence of the human TM gene was obtained by PCR amplification. The nucleotide sequence is shown in SEQ ID No. 8.

[0030] 3. The porcine TM promoter sequence, the CDS sequence of the human TM gene, and the BGHpolyA sequence (SEQ ID No. 9) were sequentially connected by enzyme ligation to construct Figure 2 Homologous recombination vectors are shown.

[0031] 4. Pig ear fibroblasts were cultured in high-glucose DMEM medium supplemented with 20% fetal bovine serum and cultured in a 5% CO2, 37°C incubator until the cells were ≥70% confluent.

[0032] 5. Mix 100 μL transfection reagent (Lonza VPI-1002, Neucleofector Kit Primary Fibroblasts) with 8 μg homologous recombination vector, digest the confluent pig ear fibroblasts with trypsin, and perform electroporation according to the NucleofectorⅡ nuclear transfection instrument program T-016. After 48 hours, the cells were cultured for monoclonal culture, and positive clones were identified by DNA level. The amplification primers are shown in Table 2.

[0033] Table 2 Amplification primers 6. The obtained positive cell clones are used for somatic cell nuclear transplantation and embryo transplantation.

[0034] 7. After the birth of a litter of cloned piglets (F0 generation) from the same cloning site, pig ear samples were cut and used for genomic DNA identification of exogenous gene integration. The results are as follows: Figure 3 As shown, 4 piglets showed integration of foreign genes.

[0035] 8. Take one of the piglets, isolate the aortic endothelial cells, establish a cell line, and identify the transgenic expression by flow cytometry. The results are as follows: Figure 4 As shown, tissues were collected for immunohistochemical identification, and the results were as follows Figure 5 As shown, significant expression of the transgene could be detected in vascular endothelial cells in all tissues.

[0036] 9. The positive cloned piglets are used for sub-generation breeding. One of the pigs with good expression is tracked and identified for its F1, F2, and F3 generation piglets. The identification results are as follows: Figure 6-8 As shown, transgene expression is Figure 9-10 As shown. From the above results, it can be seen that the transferred genes can be stably inherited and expressed consistently.

[0037] 10. The whole genome DNA of F0 generation pigs was sequenced. Through analysis and comparison of the results, it was determined that the insertion position of the transgenic expression vector was in the region between 31250963-31252424 of chromosome 11 of the pig genome. Fig.11 shown.

[0038] 2. Optimization and Verification of Safe Sites 1. After analyzing the insertion site obtained in the previous experiment, the site is located at 30963218-32221030 of pig chromosome 11, and the genomic data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5. The site is the intergenic region between tubulin alpha-1B chain-like and histoneH3.3-like. Among them, the upstream L is 1kb downstream of the tubulin alpha-1B chain-like gene. Three specific knockout gRNAs, L-SGRNA1-3, were designed by the CRISPR / Cas9 system. The sequences were synthesized by Suzhou GeneWeizhi Company. 5µg gRNA and 10µg Cas9 protein (IDT10008100, Alt-R™ Sp Cas9-GFPV3) were co-transfected into cells, and the cell genome was extracted 48 hours later for knockout efficiency identification; the downstream R, 1kb upstream of the histone H3.3-like gene, was designed by the CRISPR / Cas9 system for three specific knockout gRNAs, R-SGRNA1-3, and the sequences were synthesized by Suzhou Genewise. 5µg gRNA and 10µg Cas9 protein (IDT10008100, Alt-R™ Sp Cas9-GFP V3) were co-transfected into cells, and the cell genome was extracted 48 hours later for knockout efficiency identification. The results of knockout efficiency verification are shown in the table.

[0039] Table 3 Different sites and their knockout efficiency Subsequently, L-SGRNA3 and R-SGRNA2 with higher efficiency were selected for site-specific integration verification.

[0040] 2. Amplify the 800 bp sequence upstream and downstream of the cleavage site by PCR as the left and right homology arms, and connect the left homology arm, CBH promoter, EGFP sequence, SV40 polyA sequence, and right homology arm in sequence to construct vector L for L-SGRNA3 and vector R for R-SGRNA2, as shown in the following example. Fig.12 shown.

[0041] 3. Following the same method as the previous experiment, vector L and vector R were transfected into pig ear fibroblasts, and positive cell clones were screened for GFP expression identification. The screening results are as follows: Fig.13 The experimental results show that both sites can integrate foreign genes and the foreign genes are stably expressed.

[0042] Through the above embodiments and experimental examples, it can be seen that the present invention provides a safe site that can stably integrate exogenous genes, and has been fully verified on transgenic animals. After editing at this position, the animal can develop and reproduce normally. The site can be used for gene safety knock-in and can ensure the normal and stable expression of the transferred gene. The exogenous gene can be stably and efficiently expressed during the passage process. The safe site area reaches 2Mb, which supports the insertion of long fragments. At the same time, the safe site is very far away from the upstream and downstream genes, and the potential risk of affecting gene expression is low. Therefore, the safe site provided by the present invention has a good application prospect in genetic engineering.

Claims

1. The use of the safe site gene fragment located at 30963218-32221030 of pig chromosome 11 in the site-specific integration of foreign genes into the pig genome. The genome data are Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.

5.

2. The use according to claim 1, characterized in that: The safety site gene fragment is located at 31250963-31252424 of pig chromosome 11.

3. The use according to claim 1, characterized in that: The safety site gene fragment is the region between tubulin alpha-1B chain-like and histone H3.3-like genes.

4. The use according to any one of claims 1 to 3, characterized in that: In the safety site gene fragment, the nucleotide sequence of the site for inserting the foreign gene is shown in any one of SEQ ID NO.1-SEQ ID NO.

6.

5. A homologous recombination vector, characterized in that: Used for site-specific integration of foreign genes in the safe site gene fragment, the safe site gene fragment is located at 30963218-32221030 of pig chromosome 11, and the genome data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5; The homologous recombination vector comprises a promoter sequence, a foreign gene fragment and a transcription termination signal sequence.

6. The homologous recombination vector according to claim 5, characterized in that: The promoter sequence is shown as SEQ ID NO.7 or SEQ ID NO.12; and / or, the transcription termination signal sequence is shown as SEQ ID NO.9 or SEQ ID NO.

14.

7. The homologous recombination vector according to claim 5, characterized in that: The homologous recombination vector further comprises a left homology arm and a right homology arm.

8. The homologous recombination vector according to claim 7, characterized in that: The nucleotide sequence of the left homology arm is shown in SEQ ID NO.10, and the nucleotide sequence of the right homology arm is shown in SEQ ID NO.11; Alternatively, the nucleotide sequence of the left homology arm is as shown in SEQ ID NO.15, and the nucleotide sequence of the right homology arm is as shown in SEQ ID NO.

16.

9. A method for constructing a transgenic pig, characterized by: The foreign gene was site-specifically integrated into the safe site gene fragment, which was located at 30963218-32221030 of pig chromosome 11, and the genomic data were Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.

5.

10. The construction method according to claim 9, characterized in that: The steps include: Cultivating donor cells; transferring a homologous recombination vector for integrating foreign genes into the donor cell; Identify positive clones; The positive cell clones obtained were used for somatic cell nuclear transplantation and embryo transplantation.

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