Use of exogenous gene integration site, homologous recombination vector and construction method of transgenic pigs

Abstract

The present invention belongs to the technical field of genetic engineering, and particularly relates to the use of an exogenous gene integration site, a homologous recombination vector, and a method for constructing a transgenic pig. The safe site for exogenous gene integration provided by the present invention is the region between the genes LOC100155859 tubulin alpha-1B chain-like and LOC100517052 histone H3.3-like on porcine chromosome 11. The present invention further fully verifies this safe site in transgenic animals and cells. During the passage process, the exogenous gene can be stably and highly expressed, and has no side effects on cells and tissues. Therefore, the safe site provided by the present invention has good application prospects in genetic engineering.

Description

Technical Field

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

[0002] Currently, the methods for constructing stable cell lines and transgenic animals that are used more frequently are to integrate the target gene into a genomic safe site or randomly integrate it, and then obtain the required recombinant cell lines through a large number of screenings. One of the difficulties of this method lies in the variability and uncertainty of the random integration sites of the target gene, which may lead to phenomena such as the loss of the target gene copy number and gene rearrangement during the long-term culture of the recombinant cell line, thereby affecting the expression stability of the cells in the later stage.

[0003] Here, the concept of position effect is proposed. The position effect refers to the direct relationship between the integration site of the exogenous target gene and the cell line characteristics (such as the expression level of the exogenous target gene and the stability of the recombinant cells). Related research points out that the position effect can have enhancing and weakening effects on the expression of the exogenous target gene. Different gene integration sites have different structures and regulatory backgrounds, and the gene expression active regions are generally located in the chromosomal open regions. Therefore, affected by different integration sites, the expression levels and stabilities of the recombinant cell lines in the long-term culture are not the same.

[0004] In order to ensure the safety and normal expression of the exogenous gene insertion, developing genomic safe harbors (GSHs) on the animal genome is an important task. The safe sites provide a safe and reliable docking "harbor" for the exogenous gene, enabling the exogenous gene to be stably and highly expressed, and having no side effects on cell growth, embryonic development, and individual health. Existing research has discovered multiple safe sites that can be used for the insertion of mammalian exogenous genes (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, the H11 and AAVS1 sites have also been used as pig genomic safe sites.

[0005] However, the expression level of exogenous genes is closely related to the insertion site. Studies have found that insertion sites within certain genes cannot enable high-level expression of exogenous genes; after inserting exogenous genes at certain gene loci (such as GGTA1 and CMAH), effective expression cannot be obtained; gene expression changes occur during cell passage at some so-called "safe sites", resulting in insufficient stability. For example, it has been found that there are significant variations in the expression and silencing of a reporter gene inserted at the AAVS1 safe site among different human induced pluripotent stem cell (hiPSC) clones, which seriously affects the stability of gene expression. In addition, the regions of some safe sites are relatively narrow, making it difficult to insert large fragment vectors. For example, the H11 site is located between 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. Moreover, the safe site is relatively close to the upstream and downstream genes, which may potentially affect the expression of adjacent genes.

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

[0007] 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] Use of a safe site gene fragment located at 30963218 - 32221030 on porcine chromosome 11 in the site-directed integration of exogenous genes into the porcine genome, and the genomic data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5.

[0009] Preferably, the safe site gene fragment is located at 31250963 - 31252424 on porcine chromosome 11.

[0010] Preferably, the safe site gene fragment is the region between the tubulin alpha-1B chain-like and histone H3.3-like genes.

[0011] Preferably, in the safe site gene fragment, the nucleotide sequence of the site for inserting an exogenous gene is shown as any one of SEQ ID NO.1 - SEQ ID NO.6.

[0012] The present invention also provides a homologous recombination vector for site-specific integration of an exogenous gene into a safe-site gene fragment, where the safe-site gene fragment is located at positions 30963218 - 32221030 on porcine chromosome 11, and the genomic data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5;

[0013] The homologous recombination vector includes a promoter sequence, an exogenous gene fragment, and a transcription termination signal sequence.

[0014] Preferably, the promoter sequence is as shown in SEQ ID NO.7 or SEQ ID NO.12; and / or, the transcription termination signal sequence is as shown in SEQ ID NO.9 or SEQ ID NO.14.

[0015] Preferably, the homologous recombination vector further includes a left homologous arm and a right homologous arm.

[0016] Preferably, the nucleotide sequence of the left homologous arm is as shown in SEQ ID NO.10, and the nucleotide sequence of the right homologous arm is as shown in SEQ ID NO.11;

[0017] or, the nucleotide sequence of the left homologous arm is as shown in SEQ ID NO.15, and the nucleotide sequence of the right homologous arm is as shown in SEQ ID NO.16.

[0018] The present invention also provides a method for constructing a transgenic pig, which site-specifically integrates an exogenous gene into a safe-site gene fragment, where the safe-site gene fragment is located at positions 30963218 - 32221030 on porcine chromosome 11, and the genomic data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5.

[0019] Preferably, it includes the following steps:

[0020] Cultivate donor cells;

[0021] Transfer the homologous recombination vector for integrating the exogenous gene into the donor cells;

[0022] Identify positive clones;

[0023] Use the obtained positive cell clones for somatic cell nuclear transfer and embryo transfer.

[0024] The present invention provides a safe site for stable integration of foreign genes in the genome of pigs. After verification in transgenic animals, after inserting foreign genes into the safe site provided by the present invention, during the process of passing on generations, the foreign genes can be stably and highly expressed, and have no side effects on cells and tissues. In addition, the safe site region reaches 2 Mb, supports the insertion of long fragments, and the safe site is very far from the upstream and downstream genes, with a relatively low potential risk of affecting gene expression. Therefore, the safe site provided by the present invention has good application prospects in genetic engineering.

[0025] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0026] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0027] Figure 1 It is a schematic diagram of the regional position between the genes LOC100155859 tubulin alpha-1B chain-like and LOC100517052 histone H3.3-like on chromosome 11 of pigs;

[0028] Figure 2 It is a construction diagram of the homologous recombination vector in Experimental Example 1;

[0029] Figure 3 It is the detection result of foreign gene expression in F0 generation cloned piglets in Experimental Example 1;

[0030] Figure 4 It is the result of flow cytometry identification of transgenic expression in F0 generation cloned piglets in Experimental Example 1;

[0031] Figure 5 It is the experimental result of detecting transgenic expression in vascular endothelial cells of F0 generation cloned piglets in Experimental Example 1;

[0032] Figure 6 It is the detection result of foreign gene expression in F1 generation piglets in Experimental Example 1;

[0033] Figure 7 It is the detection result of foreign gene expression in F2 generation piglets in Experimental Example 1;

[0034] Figure 8 It is the detection result of foreign gene expression in F3 generation piglets in Experimental Example 1;

[0035] Figure 9 Experimental results of transgenic expression in piglets of F1-F3 generations in Experimental Example 1

[0036] Figure 10 Targeting results of transgenic expression in piglets of F1-F3 generations in Experimental Example 1

[0037] Figure 11 Schematic diagram of the insertion position of the transgenic expression vector in Experimental Example 1

[0038] Figure 12 Construction diagrams of vector L and vector R in Experimental Example 1

[0039] Figure 13 Detection results of exogenous gene expression after transfection of vector L and vector R into porcine ear fibroblasts respectively in Experimental Example 1 Specific implementation manners

[0040] In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available products

[0041] In the examples, the sequences involved are as follows

[0042] Table 1 Some sequences involved in the examples

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] Example 1 Safe site and homologous recombination vector for site-directed integration of exogenous genes into the porcine genome

[0053] This embodiment provides a safe site in the pig genome that can be used for site-directed integration of foreign genes. It is the region between the LOC100155859 tubulin alpha-1B chain-like gene and the LOC100517052 histone H3.3-like gene on pig chromosome 11 (Sus scrofa chromosome 11, 30963218-32221030), such as Figure 1 the blue box part shown in

[0054] As a preferred solution, this safe site is located at Sus scrofa chromosome 11, 31250963-31252424.

[0055] The homologous recombination vector for inserting foreign genes at this safe site includes a promoter sequence, a foreign gene fragment, and a transcription termination signal sequence. As a preferred solution, the homologous recombination vector includes a left homologous arm, a promoter sequence, a foreign gene fragment, a transcription termination signal sequence, and a right homologous arm.

[0056] The method for constructing transgenic pigs based on this safe site includes the following steps:

[0057] Construct a homologous recombination vector, transfect donor cells with this vector, obtain positive cell clones through DNA and RNA identification, then use these cells for somatic cell cloning. After obtaining cloned piglets, conduct DNA and protein expression identification on the piglets, and screen high-expression piglets for breeding and passaging.

[0058] The technical solution of the present invention will be further described through experiments below.

[0059] Experimental Example 1 Verification of the Safe Site

[0060] This experimental example verified the safe site provided in Example 1. The specific experimental process is as follows:

[0061] I. Determine the location of the safe site

[0062] 1. Construction of the vector for inserting foreign genes

[0063] By PCR method, the pig TM promoter sequence was amplified from the pig genome. Its nucleotide sequence is shown in SEQ ID No. 7 and is used to drive the expression of foreign genes.

[0064] 2. By PCR method, extract the DNA of the human aortic endothelial cell line, and PCR amplify the CDS sequence of the human TM gene. Its nucleotide sequence is shown in SEQ ID No. 8.

[0065] 3. Sequentially ligate the porcine TM promoter sequence, the CDS sequence of the human TM gene, and the BGH polyA sequence (SEQ ID No. 9) by restriction enzyme digestion and ligation to construct Figure 2 the homologous recombination vector shown below.

[0066] 4. Porcine 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 reached ≥ 70% confluence.

[0067] 5. Mix 100 μL of transfection reagent (Lonza VPI-1002, Neucleofector Kit Primary Fibroblasts) with 8 μg of homologous recombination vector, digest the confluent porcine ear fibroblasts with trypsin, and perform electroshock according to the Nucleofector Ⅱ nucleofector program T-016. After 48 h, the cells were subjected to monoclonal culture, and positive clones were identified at the DNA level. The amplification primers are shown in Table 2.

[0068] Table 2 Amplification primers

[0069]

[0070] 6. The obtained positive cell clones were used for somatic cell nuclear transfer and embryo transfer.

[0071] 7. After a litter of cloned piglets (F0 generation) from the same clone point were born, pig ear samples were taken for genomic DNA identification of foreign gene integration. The results are shown Figure 3 below, and 4 piglets showed foreign gene integration.

[0072] 8. Take one of the piglets, isolate aortic endothelial cells to establish a cell line, and identify the transgenic expression by flow cytometry. The results are shown Figure 4 below. Collect tissues for immunohistochemical identification. The results are shown Figure 5 below, and obvious transgenic expression could be detected in vascular endothelial cells in each tissue.

[0073] 9. The positive cloned piglets were used for subculture and breeding. One pig with good expression was tracked and identified for its F1, F2, and F3 generation piglets. The identification results are shown Figures 6 - 8 below, and the transgenic expression is shown Figures 9 - 10 below. From the above results, it can be seen that the transferred gene can be stably inherited and expressed consistently.

[0074] 10. Perform whole-genome DNA sequencing on the F0 generation pigs. Through result analysis and comparison, it was determined that the insertion position of the transgenic expression vector was in the region between 31250963 and 31252424 on chromosome 11 of the porcine genome, as shown Figure 11 below.

[0075] II. Optimization and Verification of Safe Sites

[0076] 1. After analyzing the insertion sites obtained from the previous experiment, the site is located at 30963218 - 32221030 on Sus scrofa chromosome 11, and the genomic data is Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5. This site is the intergenic region between tubulin alpha-1B chain-like and histone H3.3-like genes. Among them, the upstream L is 1 kb downstream of the tubulin alpha-1B chain-like gene. Three specific knockout gRNAs, L-SGRNA1-3, were designed through the CRISPR / Cas9 system, and the sequences were synthesized by Genewiz Suzhou. 5 μg of gRNA and 10 μg of Cas9 protein (IDT10008100, Alt-R™ S.p. Cas9-GFPV3) were co-transfected into cells, and the cell genome was extracted after 48 h for knockout efficiency identification; the downstream R is 1 kb upstream of the histone H3.3-like gene. Three specific knockout gRNAs, R-SGRNA1-3, were designed through the CRISPR / Cas9 system, and the sequences were synthesized by Genewiz Suzhou. 5 μg of gRNA and 10 μg of Cas9 protein (IDT10008100, Alt-R™ S.p. Cas9-GFP V3) were co-transfected into cells, and the cell genome was extracted after 48 h for knockout efficiency identification. The results of verifying the knockout efficiency are shown in the table below.

[0077] Table 3 Different Sites and Their Knockout Efficiencies

[0078]

[0079] Subsequently, L-SGRNA3 and R-SGRNA2 with higher efficiency were selected for site-directed integration verification.

[0080] 2. The 800-bp sequences upstream and downstream of the cleavage site were amplified by PCR as the left and right homologous arms, and the left homologous arm, CBH promoter, EGFP sequence, SV40 polyA sequence, and right homologous arm were connected in sequence to construct vector L targeting L-SGRNA3 and vector R targeting R-SGRNA2, as Figure 12 shown.

[0081] 3. According to the same method as in the previous experiment, after transfecting vector L and vector R into porcine ear fibroblasts respectively, positive cell clones were screened and GFP expression was identified. The screening results are as Figure 13As shown. The experimental results show that both of these two sites can integrate foreign genes and the foreign genes are stably expressed.

[0082] From the above examples and experimental examples, it can be seen that the present invention provides a safe site that can stably integrate foreign genes and has been fully verified in transgenic animals. After editing at this position, the animals can develop and reproduce normally. This site can be used for safe gene knock-in and can ensure the normal and stable expression of the transferred genes. During the process of passing on generations, the foreign genes can be stably and highly expressed. The region of this safe site reaches 2 Mb, supporting the insertion of long fragments. At the same time, this safe site is very far from the upstream and downstream genes, and the potential risk of affecting gene expression is relatively low. Therefore, the safe site provided by the present invention has good application prospects in genetic engineering.

Claims

1. Use of the safe-site gene fragment located at 31250963-31252424 on porcine chromosome 11 in site-directed integration of foreign genes into the porcine genome, with genomic data Sscrofa11.1, Chromosome 11, GCF_000003025.6, NC_010453.5.

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