A method of constructing a pig endogenous retrovirus-inactivated xenotransplant donor pig
By designing specific sgRNAs in porcine fetal fibroblasts and combining them with a multi-round screening strategy, and using CRISPR/Cas9 gene editing technology, the complete knockout of the porcine endogenous retrovirus PERV-pol gene was achieved. This solves the problem of the difficulty in efficiently removing PERVs in existing technologies and improves the safety of xenotransplantation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YUNNAN AGRICULTURAL UNIVERSITY
- Filing Date
- 2025-09-24
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient to efficiently remove porcine endogenous retroviruses (PERVs), especially gene editing techniques, which cannot completely knock out PERVs, leading to a high risk of infection in xenotransplantation.
Specific sgRNA sequences were designed in porcine fetal fibroblasts using CRISPR/Cas9 gene editing technology. Combined with a multi-round screening strategy, the PERV-pol gene was gradually knocked out through somatic cell cloning and embryo transfer. PCR and deep sequencing were used for initial screening and identification.
The complete knockout of the porcine endogenous retrovirus PERV-pol gene was achieved, reducing the risk of infection in xenotransplantation and improving the safety and reliability of xenotransplantation donor pigs.
Smart Images
Figure CN120866423B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of xenotransplantation technology, specifically, it relates to a method for constructing porcine endogenous retrovirus-inactivated xenotransplantation donor pigs. Background Technology
[0002] Porcine endogenous retroviruses (PERVs) are a class of retroviruses integrated into the pig genome, belonging to the pig's own genetic material rather than being exogenous infectious viruses. They possess unique biological characteristics, forming a symbiotic relationship with pigs through integration into the pig genome. Currently, the World Health Organization, in its consultations on the risks of pathogenic microorganism infection in xenotransplantation, has explicitly listed PERV infection as a crucial consideration for the clinical application of xenotransplant organs. However, PERV copy numbers in pigs are extremely high, typically ranging from 20 to 100 copies, making them impossible to eliminate using conventional methods. They can only be knocked out using gene editing technology, and completely inactivating all PERV copies is extremely difficult.
[0003] With the development of gene editing technology, cloned pigs with all PERVs copies inactivated were obtained in 2017 using CRISPR / Cas9 and somatic cell cloning technology. However, this process requires the addition of various cytokines to achieve PERVs-inactivated positive cell lines, which is technically challenging and inefficient. A method for cloning pigs with porcine endogenous retrovirus inactivation (patent number: CN201910497796.9) applied for in 2019 aims to construct PERVs knockout cloned pigs using PiggyBac transposon technology and sequential cyclic construction. However, it ultimately only obtained a partial PERV-pol gene knockout cell line, with a knockout rate of extremely low, not exceeding 20%.
[0004] Therefore, in view of the problems in the existing technology, there is an urgent need for a method to inactivate PERVs, so as to achieve the goal of efficient inactivation of PERVs, which is of great significance to promoting the development of xenotransplantation. Summary of the Invention
[0005] To address the above problems, this invention provides a method for constructing porcine endogenous retrovirus-inactivated xenotransplantation donor pigs. Based on porcine fetal fibroblast cell lines, this method utilizes CRISPR / Cas9 gene editing technology to design and synthesize a specific sgRNA sequence at the pol gene location in the transcriptional control region of porcine endogenous retroviruses. After co-incubation with spCas9 protein, the sequence is transfected into porcine fetal fibroblasts to knock out the pol gene controlling porcine endogenous retrovirus synthesis. PCR and Sanger sequencing are used for initial screening to obtain pol gene knockout cell lines. This is then organically combined with somatic cell cloning technology to obtain PERV-pol gene knockout cloned fetuses. These fetuses are then subjected to second-generation P... CR deep sequencing analysis and ddPCR determination of the remaining copy number were used. If the PERV-pol gene of the cloned fetus was completely knocked out, somatic cell cloning and embryo transfer were performed directly. If it was not completely knocked out, a secondary design and synthesis of sgRNA targeting sequences was performed and transfected into the fetal fibroblast cell line that was not completely knocked out to obtain a cell line with completely knocked-out PERVs. After somatic cell cloning and embryo transfer, PERVs-inactivated xenotransplant donor pigs were obtained. This method overcame the technical bottleneck of low efficiency of complete knockout of the porcine PERV-pol gene due to continuous drug screening and the difficulty in proliferation and survival of completely knocked-out cells. It also solved the safety problem of porcine endogenous retrovirus infection in cross-species xenotransplantation.
[0006] To achieve the above objectives, the present invention provides a method for constructing porcine endogenous retrovirus-inactivated xenotransplantation donor pigs, the method comprising the following steps:
[0007] 1) Determine the copy number of PERVs and the pol genotype;
[0008] 2) Based on the porcine fetal fibroblast cell line, an sgRNA targeting sequence was designed to knock out the PERV-pol gene by targeting the pol gene, which controls the reverse transcription of porcine endogenous retrovirus genes.
[0009] The sgRNA targeting sequences of the PERV-pol gene include PERV-sgRNA1, PERV-sgRNA2, PERV-sgRNA3, PERV-sgRNA4 and PERV-sgRNA5;
[0010] 3) The spCas9 protein and the sgRNA of the PERV-pol gene from step 2) were co-transfected into wild-type pig fetal fibroblast cell lines. The PERV-pol gene knockout cell lines were obtained by single-cell clonal culture and PCR and Sanger sequencing.
[0011] 4) The PERV-pol gene knockout cell line obtained in step 3) is subjected to somatic cell cloning and embryo transfer. After pregnancy, the fetus is removed, and the PERV-pol gene knockout pig fetal fibroblast cell line is isolated and cultured. After identifying the genotype and copy number of the PERV-pol gene, somatic cell cloning and embryo transfer are performed. After the piglets are born, they become xenotransplantation donor pigs with inactivated porcine endogenous retrovirus.
[0012] Furthermore, when the identification result in step 4) is complete knockout, the PERV-pol gene knockout pig fetal fibroblast cell line obtained in step 4) is directly used for somatic cell cloning and embryo transfer.
[0013] Furthermore, when the identification result in step 4) is that the PERV-pol gene is not completely knocked out, the non-knocked PERV-pol genotype is analyzed by second-generation PCR deep sequencing, and an sgRNA targeting sequence is designed and synthesized. This sequence is then co-transfected with spCas9 protein into the PERV-pol gene knockout pig fetal fibroblast cell line to screen for PERVs inactivation cell lines.
[0014] Furthermore, the sgRNA targeting sequences in step 4) include: PERV-sgRNA6, PERV-sgRNA7, PERV-sgRNA8 and PERV-sgRNA9; their nucleotide sequences are shown in SEQ ID NO: 12-15.
[0015] Furthermore, in step 1), the copy number of PERVs is determined by ddPCR, and the PERV-pol genotype is determined by Sanger sequencing and / or second-generation PCR deep sequencing.
[0016] The primer nucleotide sequences for the ddPCR are shown in SEQ ID NO: 1-6; the primer nucleotide sequences for the Sanger sequencing and / or second-generation PCR deep sequencing are shown in SEQ ID NO: 1 and 2.
[0017] Furthermore, in step 2), the gene targeting site for inactivating porcine endogenous retrovirus is the pol gene.
[0018] Furthermore, in step 3), the initial screening was performed using PCR and Sanger sequencing.
[0019] Further, the nucleotide sequence of PERV-sgRNA1 in step 2) is shown in SEQ ID NO: 7; the nucleotide sequence of PERV-sgRNA2 is shown in SEQ ID NO: 8; the nucleotide sequence of PERV-sgRNA3 is shown in SEQ ID NO: 9; the nucleotide sequence of PERV-sgRNA4 is shown in SEQ ID NO: 10; and the nucleotide sequence of PERV-sgRNA5 is shown in SEQ ID NO: 11.
[0020] Furthermore, the primer nucleotide sequences for the PCR and Sanger sequencing are shown in SEQ ID NO: 1 and 2.
[0021] The technical solution provided in this application may include the following beneficial effects:
[0022] This invention provides a method for constructing PERV-inactivated xenotransplantation donor pigs. Based on porcine fetal fibroblast cell lines, and using the RNP system based on CRISPR / Cas9 gene editing technology, a specific sgRNA sequence is designed and specifically cleaved at the pol gene location in the transcriptional control region of porcine endogenous retrovirus. Through a multi-round screening strategy combining low-throughput and high-throughput sequencing, the pol gene controlling the reverse transcription function of porcine endogenous retrovirus is completely knocked out, solving the problem of low knockout rate of porcine endogenous retrovirus PERV-pol gene and obtaining PERV-inactivated xenotransplantation donor pigs.
[0023] The method provided by this invention does not require the addition of any chemical inducers to maintain cell growth. Instead, it ensures cell growth activity by obtaining a fetus, making it easier to operate and implement, and it does not cause DNA damage caused by the addition of inducers.
[0024] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description
[0025] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments of this application taken in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of this application.
[0026] Figure 1 This is a flowchart of the specific implementation plan of this application.
[0027] Figure 2 The genotypes of the PERV-pol gene target region in porcine fibroblasts were analyzed using Sanger sequencing. The red markings represent the nucleotide polymorphisms (SNPs) in the target region detected by Sanger sequencing.
[0028] Figure 3 This method uses ddPCR to analyze the copy number of the porcine PERV-pol gene. Each dot represents a droplet, with the number of positive droplets above the red line and the number of negative droplets below the red line.
[0029] Figure 4 This method uses ddPCR to analyze the copy number of the porcine GAPDH gene. Each dot represents a droplet, with the number of positive droplets above the red line and the number of negative droplets below the red line.
[0030] Figure 5 The copy number of porcine PERVs was analyzed using ddPCR. The copy number was calculated as (number of PERV-pol gene-positive droplets / number of GAPDH gene-positive droplets * 2), and the highest calculated copy number was set as 46 copies for three replicates.
[0031] Figure 6 This is the Sanger sequencing result of a single porcine colony (C26). The red dashed line indicates a base deletion in this region, proving that it is a single-cell colony with the PERV-pol gene knocked out.
[0032] Figure 7 It is a pig PERV-Pol gene knockout cloned fetus.
[0033] Figure 8 The knockout rate of the PERV-pol gene in cloned fetuses with PERVs knockout was analyzed using second-generation PCR deep sequencing, and the results showed 100% knockout.
[0034] Figure 9 The analysis of the genotype of the PERV-pol gene target region was performed using Sanger sequencing. The red markings indicate the nucleotide polymorphisms (SNPs) in the target region detected by Sanger sequencing.
[0035] Figure 10 The genotype of the PERV-pol gene target region was analyzed using second-generation PCR deep sequencing. Different colors represent nucleotide polymorphisms in the region, which is largely consistent with the Sanger sequencing results.
[0036] Figure 11 This method uses ddPCR to analyze the copy number of the porcine PERV-pol gene. Each dot represents a droplet, with the number of positive droplets above the red line and the number of negative droplets below the red line.
[0037] Figure 12 This method uses ddPCR to analyze the copy number of the porcine GAPDH gene. Each dot represents a droplet, with the number of positive droplets above the red line and the number of negative droplets below the red line.
[0038] Figure 13The copy number of porcine PERVs was analyzed using ddPCR. The copy number was calculated as (number of PERV-pol gene-positive droplets / number of GAPDH gene-positive droplets * 2), and the highest calculated copy number was set as 43 copies for three replicates.
[0039] Figure 14 These are single-cell colonies obtained after cell transfection and culture, identified using PCR. The red text indicates single-cell colonies selected for Sanger sequencing.
[0040] Figure 15 This is the Sanger sequencing result of the target region of the PERV-pol gene knockout single-cell colony (C13). The red dashed line indicates the base deletion in this region, and the red background bases indicate SNP sites, proving that this single-cell colony is a PERV-pol gene knockout single-cell colony.
[0041] Figure 16 These are eight cloned fetuses obtained through somatic cell cloning using C13 single-cell colonies.
[0042] Figure 17 Eight cloned fetuses were identified using PCR. F01-F08 all showed more than two distinct bands, indicating that all eight fetuses were PERV-pol gene knockout fetuses. M is a DNA marker; WT is the control group (WildType).
[0043] Figure 18 These are Sanger sequencing results of the PERV-pol gene target region in cloned fetus F01.
[0044] Figure 19 The method uses ddPCR to analyze the PERV copy number in PERV-pol gene knockout fetuses (F01). Each dot represents a droplet, with the number of positive droplets above the red line and the number of negative droplets below the red line.
[0045] Figure 20 The method uses ddPCR to analyze the GAPDH copy number in PERV-pol gene knockout fetuses (F01). Each dot represents a droplet, with the number of positive droplets above the red line and the number of negative droplets below the red line.
[0046] Figure 21 The remaining PERV copy number of cloned fetus F01 was determined by ddPCR analysis. The copy number was calculated as (PERV-pol gene positive droplet number / GAPDH gene positive droplet number * 2), and the highest calculated copy number from three replicates was set as 7 copies.
[0047] Figure 22 The PERV-pol gene knockout rate was analyzed using second-generation PCR deep sequencing.
[0048] Figure 23 These are the PCR identification results of the second round of PERV-pol gene knockout single-cell colonies.
[0049] Figure 24 This is the result of second-generation PCR deep sequencing genotype identification of the second-round PERV-pol gene knockout single-cell colonies.
[0050] Figure 25 The method utilizes second-generation PCR deep sequencing to analyze the PERV-pol gene knockout rate in the second round of single-cell colonies.
[0051] Figure 26 This refers to the PERV-pol gene knockout performed using PiggyBac transposon technology. Detailed Implementation
[0052] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.
[0053] In preliminary research, PiggyBac transposon technology was used to sequentially construct PERVs knockout clones, but ultimately, no PERVs knockout cell lines were obtained. This was because, in 2019 (patent application number: ZL201910497796.9), whole-genome sequencing alignment was used to determine the chromosomal location of porcine endogenous retroviruses in the porcine fetal fibroblast genome. However, the retroviruses had too many repetitive sequences and too few sequencing reads, making it impossible to determine their chromosomal location using this method. After constructing an inactivated porcine endogenous retrovirus vector using PiggyBac technology and transfecting it into porcine fetal fibroblasts, cyclic screening was performed, but due to low editing efficiency, no PERV-pol gene knockout cell lines were obtained.
[0054] Because the pol gene in PERVs exhibits single nucleotide polymorphisms, meaning the sequence of each pol gene is not entirely identical, existing software cannot obtain the correct sgRNA sequence. Instead, it is necessary to determine the genotype of the pol gene in all PERVs copies of the specific individual requiring editing, and then design specific sgRNAs based on these genotypes to completely cover all PERVs copies and achieve complete PERVs inactivation.
[0055] Therefore, to address the aforementioned problems encountered during the experiment, the technology was improved, resulting in the following experimental protocol. The wild-type pig used in the protocol is the Yunnan Small-eared Pig, which has not undergone gene editing.
[0056] Example 1
[0057] A method for constructing porcine endogenous retrovirus-inactivated xenotransplantation donor pigs, the specific operation process is as follows:
[0058] 1) Determine the copy number and PERV-pol genotype of porcine endogenous retrovirus.
[0059] The PERV-pol genotype was determined using Sanger sequencing. Figure 2 The copy number of porcine endogenous retroviruses was determined by droplet digital PCR (ddPCR). Figure 3-5 ).
[0060] The primers for ddPCR include Primerpol1-FW, Primerpol1-RV, Probepol, Primerpig_GAPDH_F, Primerpig_GAPDH_R, and Probepig_GAPDH, and their nucleotide sequences are shown in SEQ ID NO: 1-6. The primers for second-generation PCR deep sequencing PERV-pol genotype sequencing include Primerpol1-FW and Primerpol1-RV, and their nucleotide sequences are shown in SEQ ID NO: 1-2.
[0061] 2) Design a targeting sequence for the pol gene, which controls the reverse transcription function of porcine endogenous retroviruses.
[0062] Based on the CRISPR / Cas9 gene editing RNP system, sgRNA targeting sequences including PERV-sgRNA1, PERV-sgRNA2, PERV-sgRNA3, PERV-sgRNA4 and PERV-sgRNA5 were designed to target the pol gene sequence that controls the reverse transcription function of porcine endogenous retroviruses. Their nucleotide sequences are shown in SEQ ID NO: 7-11. The pol gene that controls the transcription of porcine endogenous retroviruses was knocked out.
[0063] 3) Transfection screening of porcine endogenous retrovirus-inactivated monoclonal cell lines
[0064] spCas9 protein (purchased from ThermoFisher SCIENTIFIC; catalog number: A50577) and the aforementioned sgRNA targeting sequence (its nucleotide sequence is shown in SEQ ID NO: 7-11) were co-transfected into porcine fetal fibroblast cell lines via RNP (ribonucleoprotein) transfection (transfection system shown in Table 1; transfection system volume: 10 μL; transfection program: 1650V, 10ms, 3 pulses; transfection equipment: Neon Transfection system). Single-cell clones were obtained through single-cell clonal culture, and PERV-pol gene knockout cell lines were initially screened by PCR and Sanger sequencing. Figure 6 The primers used for PCR and Sanger sequencing were Primerpol1-FW and Primerpol1-RV, with nucleotide sequences shown in SEQ ID NO: 1 and 2. PERV-pol gene knockout cell lines were further screened using next-generation PCR deep sequencing.
[0065] Table 1 Transfection System
[0066]
[0067] 4) Somatic cell cloning and embryo transfer
[0068] Using somatic cell cloning technology, PERV-pol genotype knockout cells obtained through screening are cloned into somatic cells, and the fetus is retrieved upon successful pregnancy. Figure 7 ) and identified the PERV-pol gene knockout status, showing that the PERV-Pol knockout rate was nearly 100% ( Figure 8 (Table 2) confirmed that the porcine endogenous retrovirus had been completely inactivated.
[0069] Table 2 Results of a single knockout
[0070]
[0071] Example 2
[0072] A method for constructing porcine endogenous retrovirus-inactivated xenotransplantation donor pigs, the specific operation process is as follows:
[0073] 1) Determine the copy number and PERV-pol genotype of porcine endogenous retrovirus.
[0074] Using Sanger sequencing ( Figure 9 ) and second-generation PCR depth ( Figure 10Sequencing was used to determine the PERV-pol genotype, and the copy number of porcine endogenous retrovirus was determined by droplet digital PCR (ddPCR). Figure 11-13 ).
[0075] The primers for ddPCR include Primerpol1-FW, Primerpol1-RV, Probepol, Primerpig_GAPDH_F, Primerpig_GAPDH_R, and Probepig_GAPDH, and their nucleotide sequences are shown in SEQ ID NO: 1-6. The primers for second-generation PCR deep sequencing PERV-pol genotype sequencing include Primerpol1-FW and Primerpol1-RV, and their nucleotide sequences are shown in SEQ ID NO: 1-2.
[0076] 2) Design a targeting sequence for the pol gene, which controls the transcription of porcine endogenous retrovirus genes.
[0077] Based on the porcine fetal fibroblast cell line, and using the CRISPR / Cas9 gene editing RNP system, sgRNA targeting sequences including PERV-sgRNA1, PERV-sgRNA2, PERV-sgRNA3, PERV-sgRNA4, and PERV-sgRNA5 were designed to target the pol gene sequence that controls the transcription of porcine endogenous retroviruses. Their nucleotide sequences are shown in SEQ ID NO: 7-11. The pol gene that controls the transcription of porcine endogenous retroviruses was knocked out.
[0078] 3) Transfection screening of porcine endogenous retrovirus-inactivated monoclonal cell lines
[0079] spCas9 protein (purchased from ThermoFisher SCIENTIFIC; catalog number: A50577) and the aforementioned sgRNA targeting sequence (its nucleotide sequence is shown in SEQ ID NO: 7-11) were co-transfected into porcine fetal fibroblast cell lines via RNP (ribonucleoprotein) (transfection system shown in Table 1; transfection system volume: 10 μL; transfection program: 1650V, 10ms, 3 pulses; transfection equipment: Neon Transfection system). Single-cell clones were obtained through single-cell clonal culture and subjected to PCR (…). Figure 14 Sanger sequencing Figure 15Initial screening yielded PERV-pol gene knockout cell lines. The primers used for PCR and Sanger sequencing were Primerpol1-FW and Primerpol1-RV, with nucleotide sequences shown in SEQ ID NO: 1 and 2. Further screening using next-generation PCR deep sequencing yielded PERV-pol gene knockout cell lines. As shown in Table 3, the knockout rate in the first round reached 93.95%.
[0080] Table 3. First round of PERV-pol gene knockout ratio
[0081]
[0082] 4) Somatic cell cloning and embryo transfer
[0083] Using conventional somatic cell cloning techniques, the selected PERV-pol genotype knockout cells were cloned into somatic cells, and the fetus was retrieved upon successful pregnancy. Figure 16 And identify the PERV-pol gene knockout status ( Figure 17-22 To determine whether porcine endogenous retroviruses are inactivated, corresponding porcine fetal fibroblast cell lines with inactivated porcine endogenous retroviruses were obtained through routine isolation and culture.
[0084] Example 3
[0085] 1) Based on the PERV-pol gene knockout results obtained in Example 2, and using the RNP system of CRISPR / Cas9 gene editing, sgRNA targeting sequences were designed for the pol gene sequence that controls the transcription of porcine endogenous retroviruses. These sequences include: PERV-sgRNA6, PERV-sgRNA7, PERV-sgRNA8, and PERV-sgRNA9. Their nucleotide sequences are shown in SEQ ID NO: 12-15.
[0086] 2) Transfection screening of porcine endogenous retrovirus-inactivated monoclonal cell lines
[0087] spCas9 protein (catalog number: A50577) and sgRNA targeting sequence (nucleotide sequence as shown in SEQ ID NO: 12-15) were co-transfected into porcine fetal fibroblast cell lines for secondary knockout (transfection system is shown in Table 4; transfection system volume: 10 μL; transfection program: 1650V, 10ms, 3 pulses; transfection device: Neon Transfection system). Single-cell clones were obtained by single-cell clonal culture, and PERV-pol gene knockout cell lines were obtained by PCR screening. Figure 23 ), further through second-generation PCR deep sequencing ( Figure 24-25The cell lines with PERV-pol gene knockout were identified. Table 5 shows that the knockout rate in the second round reached 99.73%.
[0088] Table 4 Transfection System
[0089]
[0090] Table 5. Second round of PERV-pol gene knockout ratio
[0091]
[0092] 3) Somatic cell cloning and embryo transfer
[0093] Using conventional somatic cell cloning technology, the selected PERV-pol gene-completely knocked-out cells were cloned into somatic cells, and after the piglets were born, xenotransplant donor pigs with complete knockout of porcine endogenous retroviruses were obtained.
[0094] Table 6. Sequence Correspondence Table in this Application
[0095]
[0096] By utilizing the method of completely inactivating porcine endogenous retroviruses in the examples, porcine fetal fibroblast cell lines and individuals with completely inactivated porcine endogenous retroviruses were efficiently obtained. This method overcomes the difficulties and low success rates of existing porcine endogenous retrovirus knockout techniques. It is of great research significance for reducing the risk of cross-species PERV transmission during xenotransplantation and for accelerating the development of xenotransplantation donor pigs.
[0097] Comparative Example 1
[0098] Based on a method for inactivating porcine endogenous retroviruses to create cloned pigs disclosed in 2019 (patent number: CN201910497796.9), experiments were conducted to construct PERVs knockout cloned pigs using PiggyBac transposon technology and sequential cycling. However, only partially PERVs knockout cell lines were ultimately obtained. In the experiments, ddPCR and whole-genome sequencing were used to determine the copy number of porcine endogenous retroviruses in the porcine fetal fibroblast genome and their chromosomal location, but the results were unsatisfactory; a porcine fibroblast cell line with complete knockout of endogenous retroviruses was not obtained, and the expected invention effect was not achieved.
[0099] Therefore, it can be concluded that the method used in this comparative experiment cannot obtain a porcine fibroblast cell line with complete knockout of endogenous retroviruses.
[0100] Comparative Example 2
[0101] According to previous reports on the complete inactivation of PERV, the first step is to construct a PiggyBac transposon vector that induces Cas9 and sgRNA expression with tetracycline (Dox). This vector is then transfected into porcine PK15 cells, and positive cell lines are selected. PERV-pol gene knockout cell clones are then obtained through continuous Dox addition. Following this method, the PiggyBac transposon vector, which induces Cas9 and sgRNA expression with Dox, is transfected into porcine fetal fibroblasts. The first batch of transfections is followed by Dox addition and continuous selection for 22 days; the second batch is followed by Dox addition and continuous selection for 14 and 23 days; and the third batch is followed by Dox addition and continuous selection for 7 and 17 days. The knockout rate in all cases does not exceed 20% (e.g., ...). Figure 26 (As shown).
[0102] However, because Dox causes DNA damage and mutations, it easily leads to cellular senescence and apoptosis. Therefore, multiple cytokines, such as p53 inhibitor, PFTα, fibroblast growth factor, and bFGF, need to be added to prevent cellular senescence and apoptosis, thus enabling the screening of cell lines with complete PERV-pol gene knockout. However, the p53 gene is a DNA damage repair gene; adding p53 inhibitors prevents effective DNA repair within cells, leading to adverse consequences such as genetic mutations and tumorigenesis.
[0103] This invention eliminates the need to construct tetracycline (Dox)-induced Cas9 expression cell lines, transfect with lentivirally packaged sgRNA vectors, or add various cytokines to maintain cell growth. Simply deliver the synthesized sgRNA sequence and commercially available spCas9 protein into cells via electroporation, followed by single-cell culture and screening. This allows for the selection of cell lines with complete PERV-pol gene knockout. Even if a complete PERV-pol gene knockout cell line is not obtained in the first round, it can be achieved through a second round of knockout.
[0104] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.
Claims
1. A method for constructing porcine endogenous retrovirus-inactivated xenotransplantation donor pigs, characterized in that, The method includes the following steps: 1) Determine the copy number and pol genotype of PERVs; 2) Based on the porcine fetal fibroblast cell line, an sgRNA targeting sequence was designed to knock out the PERV-pol gene by targeting the pol gene, which controls the reverse transcription function of porcine endogenous retroviruses. The sgRNA targeting sequences of the PERV-pol gene include PERV-sgRNA1, PERV-sgRNA2, PERV-sgRNA3, PERV-sgRNA4 and PERV-sgRNA5; The nucleotide sequence of PERV-sgRNA1 is shown in SEQ ID NO: 7; the nucleotide sequence of PERV-sgRNA2 is shown in SEQ ID NO: 8; the nucleotide sequence of PERV-sgRNA3 is shown in SEQ ID NO: 9; the nucleotide sequence of PERV-sgRNA4 is shown in SEQ ID NO: 10; and the nucleotide sequence of PERV-sgRNA5 is shown in SEQ ID NO:
11. 3) The spCas9 protein and the sgRNA of the PERV-pol gene from step 2) were co-transfected into wild-type pig fetal fibroblast cell lines. The PERV-pol gene knockout cell lines were obtained by single-cell clonal culture, PCR and Sanger sequencing. 4) The PERV-pol gene knockout cell line obtained in step 3) is subjected to somatic cell cloning and embryo transfer. After pregnancy, the fetus is removed, and the PERV-pol gene knockout pig fetal fibroblast cell line is isolated and cultured to identify the genotype and copy number of the PERV-pol gene. When the identification result is complete knockout, the obtained PERV-pol gene knockout pig fetal fibroblast cell line is directly used for somatic cell cloning and embryo transfer. When the identification result is incomplete knockout, the non-knockout PERV-pol genotype is determined by next-generation sequencing, and sgRNA targeting sequences are designed and synthesized. These sequences are then co-transfected with spCas9 protein into the PERV-pol gene knockout porcine fetal fibroblast cell line. After screening to obtain PERVs-inactivated cell lines, somatic cell cloning and embryo transfer are performed. The sgRNA targeting sequences include: PERV-sgRNA6, PERV-sgRNA7, PERV-sgRNA8, and PERV-sgRNA9; their nucleotide sequences are shown in SEQ ID NO: 12-15. Once the piglets are born, they become xenotransplant donor pigs with inactivated porcine endogenous retroviruses.
2. The method according to claim 1, characterized in that, In step 1), the copy number of PERVs is determined by ddPCR, and the PERV-pol genotype is determined by Sanger sequencing and / or next-generation sequencing. The nucleotide sequences of the primers for ddPCR are shown in SEQ ID NO: 1-6; the nucleotide sequences of the primers for Sanger sequencing and / or next-generation sequencing are shown in SEQ ID NO: 1 and 2.
3. The method according to claim 1, characterized in that, The nucleotide sequences of the primers for PCR and Sanger sequencing in step 3) are shown in SEQ ID NO: 1 and 2.
4. The method according to claim 1, characterized in that, The volume of the co-transfection system was 10 μL, and the transfection program was 1650 V, 10 ms, 3 pulses.