CN110373390A - A kind of construction method of xenotransplant basis donor pig - Google Patents

Abstract

The present invention relates to a kind of construction methods of xenotransplant basis donor pig, belong to Animal Biotechnology field.On the basis of fetal fibroblast, tri- genes of GGTA1, CMAH, B4GalNT2 are knocked out using CRISPR/Cas9 gene editing technology, and insertion tri- humanization modifiers of hCD55, hCD59, hCD46 are pinpointed, in conjunction with the xenotransplantation donors basis pig of somatic cell nuclear transfer technique building GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46 genes modification.The present invention solves the common problem that antigen-antibody immunological rejection present in xenotransplant and complement are adjusted to a certain extent, it is suitble to the building of the xenotransplant donor pig of the tissue specificities such as organs and pancreas islet, skin such as heart, kidney, liver to lay a good foundation to research and develop on the basis of basic donor pig, there is important application value and application prospect to the xenotransplantation donors pig of building genes modification.

Description

A kind of construction method of xenotransplant basis donor pig

Technical field

The invention belongs to Animal Biotechnology fields, specifically, being related to a kind of xenotransplant basis donor pig Construction method.

Background technique

Organ transplant is to treat the unique channel of terminal phase organ failure patient, and heterograft is to solve human organ to supply The most effective approach of body critical shortage.In recent years, with China's diabetes, chronic kidney disease, cardiovascular disease and hepatitis etc. Patients with chronic diseases increases substantially, so that the patient for developing into the organ failures such as terminal phase liver, kidney, the heart sharply increases Add, it also will be increasing to the pressure of organ demand.According to incompletely statistics, clinical organ transplant can be implemented in China every year at present Quantity be about 10,000, and wait the patient of organ transplant then to have 1,500,000, wait organ transplant and organ transplant trouble can be carried out The quantity ratio of person has reached 150:1.Moreover, China stopped using convict sentenced to death's organ as donor from 2015 comprehensively Body source, the final source that can only use the organ that citizen voluntarily contributes after dead as China's organ transplant, this certainly will lead Causing organ donor, originally the situation of worsening shortages is more acute, this is for the huge PATIENT POPULATION that the whole nation waits donor, extremely The organ that person contributes seriously is unable to meet demand.Therefore, new transplantation donor is found to solve current organ critical shortage Problem is very urgent.

Speak of xenotransplant, pig is easy to captive breeding, and organ size matches with the mankind, physiology and metabolism aspect with A possibility that mankind are extremely close, and people pig is total to illness generation is smaller, it is few to be related to dispute of ethic, and can enhance by gene modification The matching of donor organ.Therefore, it is to be most suitable for the species as xenotransplantation donors that pig, which is recognized,.By nearly over half a century Effort and exploration, screened at present nearly 30 can be effectively relieved immunology incompatibility to overcome the related of functional disorder Gene.The donor pig of several genes modification is also developed and has carried out pig and tested to the heterograft of non-human primates.

Although pig is approved as the source of human organ transplantation, transplanting pathogenic microorganism infection risk is immunized It is serious difficult and critical that incompatibility and functional disorder three major issues face pig as xenotransplant Challenge is also not enough to convert to clinical medicine.For this purpose, a kind of building of xenotransplant basal gene modification pig will be xenogenesis New approach is opened up in organ transplant research, and boosting transplanting achieves initial success, and such as knocks out α -1,3- galactoside transferase (GGTA1) gene can avoid Hyperacute immunological rejection, and knocking out porcine endogenous retrovirus (PERVs) gene can keep away The cross-species infection for exempting from virus, solves the problems, such as the bio-safety of heterograft, but the immunology incompatibility after heterograft It more needs to pay close attention to functional disorder.Therefore, a kind of building of xenotransplant basis donor pig will be to development Xenogeneic organ The research and extension application of transplanting has great importance.

Summary of the invention

In view of the above problems, the present invention provides a kind of construction method of xenotransplant basis donor pig, this method For xenotransplant, there are the common problems such as immunology incompatibility and functional disorder, construct GTKO/CMAHKO/ The heterograft basis donor pig of six gene modification of B4GalNT2KO/hCD55/hCD59/hCD46, will be really effective for exploitation It is suitble to the donor pig of various organ xenotransplantations to lay a good foundation.

In order to achieve the above objectives, the invention provides the following technical scheme:

A kind of construction method of xenotransplant basis donor pig, specific steps are as follows:

1) it is fastened in pig fibroblast while knocking out GGTA1, CMAH, B4GalNT2 gene

On the basis of pig fibroblast, using CRISPR/Cas9 gene editing technology knock out GGTA1, CMAH, Tri- genes of B4GalNT2 obtain the pig fibroblast of tri- gene knockout of GTKO/CMAHKO/B4GalNT2KO；

2) GTKO/CMAHKO/B4GalNT2KO modification pig fibroblast fasten pinpoint again insertion hCD55, hCD59, Tri- humanization modifiers of hCD46

On the basis of tri- gene modification of GTKO/CMAHKO/B4GalNT2KO, then pinpoint insertion hCD55, hCD59, hCD46 tri- A humanization modifier obtains the pig of GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46 gene modification into fibre Tie up cell line；

3) body-cell neucleus transplanting and embryo transfer

Using somatic cell nuclear transfer technique, the GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46 that screening is obtained Six gene cell systems carry out body-cell neucleus transplanting, and the modification situation of fetus identification gene is taken out when gestation, is separately cultured foundation Six gene modification xenotransplantation donors pig fibroblasts, or it is basic to xenotransplant is obtained when being born when development of fetus Donor pig individual；

4) continuous clone technology is utilized, by the heterograft basis donor pig fetus of six gene modifications of above-mentioned acquisition at fiber finer Born of the same parents are to carry out body-cell neucleus transplanting and embryo transfer, and batch obtains xenotransplant basis donor pig, to be suitble to various organs The exploitation of transplantation donor pig is prepared.

In step 3), the fetus of gestation is taken out to the editor's situation that can identify related gene, is the gene editing of next step Strategy provides heterograft basis donor pig cell system.

Beneficial effects of the present invention:

The invention discloses a kind of construction method of xenotransplant basis donor pig, this method can move to solve Xenogeneic organ The common problems such as the immunology incompatibility of the transplanting of plant and functional disorder provide base support, are suitble to difference thin to develop The research of xenotransplantation donors pig of born of the same parents, tissue, organ are laid a good foundation, and are really effectively suitble to various cells, group to exploitation Knit, the donor pig of organ xenotransplantation is of great significance, promote the research and development process of xenotransplant.

Detailed description of the invention

Fig. 1 is flow chart of the invention.

Specific embodiment

Embodiment 1

A kind of construction method of xenotransplant basis donor pig, specific operation process sequentially include the following steps:

1) it is fastened in the pig fibroblast of porcine endogenous retrovirus inactivation and knocks out GGTA1, CMAH, B4GalNT2 gene

On the basis of pig fibroblast, GGTA1, CMAH, B4GalNT2 tri- is knocked out using CRISPR/Cas9 gene technology A gene obtains the pig fibroblast that tri- gene of GTKO/CMAHKO/B4GalNT2KO knocks out simultaneously；

2) it is fastened in the pig fibroblast of GTKO/CMAHKO/B4GalNT2KO modification and pinpoints insertion hCD55/hCD59/ again Tri- humanization modifiers of hCD46

On the basis of tri- gene modification of GTKO/CMAHKO/B4GalNT2KO, then pinpoint insertion hCD55, hCD59, hCD46 tri- A humanization modifier, obtain six gene modification of GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46 pig at Fibrocyte system；

3) body-cell neucleus transplanting and embryo transfer

Using somatic cell nuclear transfer technique, the GTKO/CMAHKO/B4GalNT2KO/hCD55/ that will be obtained through transfection screening Six gene cell system of hCD59/hCD46 carries out body-cell neucleus transplanting, and the modification feelings of fetus identification gene are taken out when gestation 30 days Condition is separately cultured and establishes six gene modification xenotransplantation donors pig fibroblasts, or is born when development of fetus is to 114d Obtain xenotransplant basis donor pig individual.

Embodiment 2

A kind of construction method of xenotransplant basis donor pig, specific operation process sequentially include the following steps:

1) it is fastened in pig fibroblast while knocking out GGTA1, CMAH, B4GalNT2 gene

Existing pig fibroblast, on the basis of, using CRISPR/Cas9 gene editing technology simultaneously knock out GGTA1, Tri- genes of CMAH, B4GalNT2 screen through transfection and obtain tri- gene of GTKO/CMAHKO/B4GalNT2KO while knocking out thin Born of the same parents system carries out body-cell neucleus transplanting again, and fetus is taken out after gestation 30 days and identifies whether be positive, acquisition GTKO/CMAHKO/ Tri- gene knock-out pig fetal fibroblast cell line of B4GalNT2KO；

3) GTKO/CMAHKO/B4GalNT2KO modification pig fibroblast fasten pinpoint again insertion hCD55, hCD59, Tri- humanization modifiers of hCD46

On the basis of the porcine fetus fibroblasts system of, tetra- gene modification of GTKO/CMAHKO/B4GaINT2KO, then pinpoint slotting Enter tri- humanization modifiers of hCD55, hCD59, hCD46, is screened through transfection and obtain PERVKO/GTKO/CMAHKO/ The cell line of six gene modification of B4GalNT2KO/hCD55/hCD59/hCD46；

4) body-cell neucleus transplanting and embryo transfer

Using somatic cell nuclear transfer technique, the GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/ that above-mentioned screening is obtained Six gene cell system of hCD46 carries out body-cell neucleus transplanting, and the modification situation of fetus identification gene is taken out when gestation, is separately cultured Six gene modification xenotransplantation donors pig fibroblasts are established, or obtain xenotransplant when development of fetus to birth Basic donor pig individual.

Utilize the common problem being directed in xenotransplant in embodiment, the xenotransplant basis donor of building Pig can accelerate research and development process of the pig as xenotransplant.Meanwhile on the basis of xenotransplant basis donor pig On, different gene editing strategies can be taken to carry out target gene for different cells, tissue, organ xenotransplantation's scheme Modification greatly will be saved screening time and save the investment of experiment fees, developed to the method modified using the assortment of genes Really effective xenotransplant donor pig has great importance, and promotes the research and development process of heterograft industrialization.

Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention rather than limits, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

Claims (1)

1. a kind of construction method of xenotransplant basis donor pig, it is characterised in that: specific steps are as follows:

1) cause the gene of antigen-antibody immunological rejection in knock-out pig genome

On the basis of pig fibroblast, GGTA1, CMAH, B4GalNT2 tri- is knocked out using CRISPR/Cas9 gene technology A specific gene；

2) fixed point insertion tri- humanization modifiers of hCD55, hCD59, hCD46

On the basis of tri- gene modification of GTKO/CMAHKO/B4GalNT2KO, then pinpoint insertion hCD55, hCD59, hCD46 tri- A humanization modifier obtains what six gene of GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46 was modified jointly Pig fibroblast；

3) body-cell neucleus transplanting and embryo transfer

Using somatic cell nuclear transfer technique, the GTKO/CMAHKO/B4GalNT2KO/hCD55/ that will be obtained through transfection screening Six gene cell system of hCD59/hCD46 carries out body-cell neucleus transplanting, and the volume of fetus identification fetus related gene is taken out when gestation Situation is collected, is separately cultured and establishes six gene heterograft basis donor pig fibroblasts, or when development of fetus to when being born Obtain xenotransplant basis donor pig；

4) continuous clone technology is utilized, by the heterograft basis donor pig fetus of six gene modifications of above-mentioned acquisition at fiber finer Born of the same parents are to carry out body-cell neucleus transplanting and embryo transfer, and batch obtains xenotransplant basis donor pig, to be suitble to various organs The exploitation of transplantation donor pig is prepared.