Construction method of donor pig capable of being used for xenogeneic organ transplantation

A construction method and a technology for donor pigs, applied in the field of animal genetic engineering, can solve the problems that hinder the wide application of allogeneic organ transplantation and the shortage of organ resources.

Inactive Publication Date: 2020-11-20
WUYI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the serious shortage of organ resources has hindered the wide application of allogeneic organ transplantation. According to statistics, there are more than 6 million patients waiting for transplantation in the world, and the number of patients is still increasing. The organ supply in developed countries can only meet 15% of the needs. , about one in five patients died while waiting

Method used

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  • Construction method of donor pig capable of being used for xenogeneic organ transplantation
  • Construction method of donor pig capable of being used for xenogeneic organ transplantation
  • Construction method of donor pig capable of being used for xenogeneic organ transplantation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of GGTA1-TALENs

[0044] (1) Design of TALENs target sequences

[0045] According to the principle of TALENs target sequence design, the target sequence was designed in exon 6 of the pig GGTA1 gene (nucleotide sequence shown in SEQ ID No. 1), and two pairs of TALENs were constructed, TALENs Set1 and TALENs Set2. See the TALENs target recognition sequence. figure 1 A and B in. The lengths of the TALENs Set1 target recognition sequences were 17bp and 16bp, respectively, and the length of the spacer sequence was 16bp; the lengths of the TALENs Set2 target recognition sequences were 16bp and 16bp, respectively, and the length of the spacer sequence was 19bp.

[0046] Table 1 TALENs target sequences

[0047]

[0048] Note: The uppercase letters on both sides are the recognition sequence (L) of the left arm TALEN module and the recognition sequence (R) of the right arm TALEN module, respectively, and the lowercase letters in the middle are the spac...

Embodiment 2

[0055] Example 2 Construction of pCAG-HLA-G5-neo expression vector

[0056] The HLA-G5 gene fragment and the pCAG-neo vector were double-enzyme digested with the same endonuclease, and then ligated at 16°C overnight after the gel was recovered, and the single clones were transformed and picked for restriction enzyme digestion and sequencing identification. The total length of the constructed pCAG-HLA-G5-neo expression vector was 7395 bp, and three target bands with sizes of 4128 bp, 2169 bp and 1098 bp were obtained by double digestion with ScaI and SacI ( image 3 A); the target bands with sizes of 6022bp and 1373bp appeared after single digestion with HindIII ( image 3 B).

[0057] The two plasmids identified by enzyme digestion were sent to BGI for sequencing and identification. The sequencing results showed that both clones were correct, and No. 1 was randomly selected as the final expression vector. The expression vector was linearized with Sac I endonuclease, and the ...

Embodiment 3

[0058] Example 3 GGTA1 gene targeting PFF mediated by TALENs transfection

[0059] (1) Isolation of primary Bama pig fetal fibroblast PFF

[0060] The pig fetuses were taken out by caesarean section on the 35th day of pregnancy, soaked in PBS supplemented with 50μg / mL kanamycin, 100IU / mL penicillin, and 100g / mL streptomycin and brought back to the laboratory. Carefully peel off the fetus and afterbirth, place the fetuses in PBS (containing 100IU / mL penicillin, 100 g / mL streptomycin), first take the head, limbs, internal organs and other tissues, and put them in EP tubes for the next step of genome extraction. . The remaining fetal tissue was washed 5 times, cut into pieces, added with 10 mL of porcine primary PFFs to separate the digestion solution, and placed in a 38°C incubator for digestion for about 4 hours. Then, transfer it to a 15 mL centrifuge tube, 1000 rpm, 5 min, collect the cells, discard the supernatant, wash the cells twice with PFFs medium, and then culture wi...

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Abstract

The invention relates to the field of animal genetic engineering, in particular to a construction method of a donor pig capable of being used for xenogeneic organ transplantation. The construction method comprises the following steps: (1) on the basis of porcine fetal fibroblasts, knocking out a GGTA1 gene, and meanwhile, transferring the GGTA1 gene into a gene HLA-G5 to obtain GTKO / HLA-G5 + clonecells; and (2) carrying out somatic cell nuclear transplantation by taking the GTKO / HLA-G5 + clonal cells as nuclear donors, and performing culturing to obtain recombinant embryos, transplanting therecombinant embryos into the surrogate sows, and carrying out farrowing after pregnancy to obtain donor pigs. The gene knockout and transgenic GTKO / HLA-G5 + cloned pig with important scientific research value and application prospect is constructed, and an important research tool and a new research way are provided for xenogeneic organ transplantation.

Description

technical field [0001] The invention relates to the field of animal genetic engineering, in particular to a method for constructing a donor pig that can be used for xenogeneic organ transplantation. Background technique [0002] With the deepening of basic research and the improvement of clinical research, organ transplantation has become the first choice for the treatment of end-stage organ diseases. The development of clinical medicine and the development of immunosuppressive agents have made allogeneic organ transplantation obtain remarkable curative effect. However, the severe shortage of organ resources has hindered the wide application of allogeneic organ transplantation. According to statistics, there are currently more than 6 million patients waiting for transplantation in the world, and the number of patients is still increasing. The supply of organs in developed countries can only meet 15% of the needs. , about one in five patients died while waiting. The increas...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/85C12N5/10
CPCA01K67/0273A01K67/0276A01K67/0278A01K2227/108A01K2267/025C07K14/70539C12N9/1051C12N15/8509C12N2800/30
Inventor 周小青唐成程邹庆剑陈敏赖良学
Owner WUYI UNIV
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