CN110373389A - 一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法 - Google Patents
一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法 Download PDFInfo
- Publication number
- CN110373389A CN110373389A CN201910497785.0A CN201910497785A CN110373389A CN 110373389 A CN110373389 A CN 110373389A CN 201910497785 A CN201910497785 A CN 201910497785A CN 110373389 A CN110373389 A CN 110373389A
- Authority
- CN
- China
- Prior art keywords
- pig
- skin
- gene
- islet cells
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004153 islets of langerhan Anatomy 0.000 title claims abstract description 37
- 238000002689 xenotransplantation Methods 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 238000003780 insertion Methods 0.000 claims abstract description 17
- 230000037431 insertion Effects 0.000 claims abstract description 17
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 16
- 238000012986 modification Methods 0.000 claims abstract description 16
- 230000004048 modification Effects 0.000 claims abstract description 16
- 210000003754 fetus Anatomy 0.000 claims abstract description 15
- 239000003607 modifier Substances 0.000 claims abstract description 14
- 238000012239 gene modification Methods 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 108091033409 CRISPR Proteins 0.000 claims abstract description 12
- 238000005516 engineering process Methods 0.000 claims abstract description 8
- 238000010374 somatic cell nuclear transfer Methods 0.000 claims abstract description 7
- 101150076800 B2M gene Proteins 0.000 claims abstract description 6
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 6
- 238000010362 genome editing Methods 0.000 claims abstract description 6
- 101000856513 Homo sapiens Inactive N-acetyllactosaminide alpha-1,3-galactosyltransferase Proteins 0.000 claims abstract description 5
- 101000902205 Homo sapiens Inactive cytidine monophosphate-N-acetylneuraminic acid hydroxylase Proteins 0.000 claims abstract description 5
- 102100025509 Inactive N-acetyllactosaminide alpha-1,3-galactosyltransferase Human genes 0.000 claims abstract description 5
- 102100022247 Inactive cytidine monophosphate-N-acetylneuraminic acid hydroxylase Human genes 0.000 claims abstract description 5
- 108010068144 beta-1,4-N-acetyl-galactosaminyl transferase 2 Proteins 0.000 claims abstract description 5
- 238000012795 verification Methods 0.000 claims abstract 2
- 230000001900 immune effect Effects 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 10
- 210000001161 mammalian embryo Anatomy 0.000 claims description 10
- 238000012546 transfer Methods 0.000 claims description 10
- 238000002474 experimental method Methods 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 230000003013 cytotoxicity Effects 0.000 claims description 6
- 231100000135 cytotoxicity Toxicity 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 230000001404 mediated effect Effects 0.000 claims description 5
- 101710175243 Major antigen Proteins 0.000 claims description 4
- 101150106031 TRI gene Proteins 0.000 claims description 4
- 230000023555 blood coagulation Effects 0.000 claims description 4
- RFHAOTPXVQNOHP-UHFFFAOYSA-N fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 claims description 4
- 230000035935 pregnancy Effects 0.000 claims description 4
- 230000007969 cellular immunity Effects 0.000 claims description 3
- 230000001427 coherent effect Effects 0.000 claims description 3
- 230000024203 complement activation Effects 0.000 claims description 3
- 230000000295 complement effect Effects 0.000 claims description 3
- 230000001744 histochemical effect Effects 0.000 claims description 3
- 210000004698 lymphocyte Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000002054 transplantation Methods 0.000 abstract description 12
- 241001465754 Metazoa Species 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 206010053567 Coagulopathies Diseases 0.000 description 6
- 208000015294 blood coagulation disease Diseases 0.000 description 6
- 230000009852 coagulant defect Effects 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 210000000496 pancreas Anatomy 0.000 description 5
- 206010061218 Inflammation Diseases 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 230000006058 immune tolerance Effects 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000005034 decoration Methods 0.000 description 3
- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 201000001880 Sexual dysfunction Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 230000004203 pancreatic function Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 231100000872 sexual dysfunction Toxicity 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12N15/877—Techniques for producing new mammalian cloned embryos
- C12N15/8778—Swine embryos
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
- A01K2267/025—Animal producing cells or organs for transplantation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Plant Pathology (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明涉及一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,属动物生物技术领域。在猪胎儿成纤维细胞的基础上,利用CRISPR/Cas9基因编辑技术敲除GGTA1、CMAH、B4GalNT2三个基因并定点插入hCD55、hCD59、hCD46三个人源化修饰基因,再结合体细胞核移植技术构建上述六基因修饰的异种移植供体的基础猪,在此基础猪上再敲除B2M基因,同时定点插入hCD47、A20、CTLA4‑Ig三个基因,再利用体细胞核移植技术构建多基因修饰的胰岛细胞及皮肤异种移植供体猪,再对该供体猪进行功能验证,获得适合胰岛细胞及皮肤异种移植供体猪。本发明为异种胰岛细胞及皮肤等移植供体猪的开发提供了全新的研究思路和方法,促进了猪作为异种胰岛细胞及皮肤移植的临床转化和应用。
Description
技术领域
本发明属于动物生物技术领域,具体的说,涉及一种适合胰岛细胞及皮肤组织异种移植供体猪的构建方法。
背景技术
胰岛细胞移植技术对于胰腺功能完全衰竭的糖尿病患者而言,是一个行之有效的重要途径。当前胰岛的分离技术已经较成熟,但免疫排斥和移植功能障碍的问题尚未得到很好的解决,进而导致该技术没有被广泛的推广应用。
谈到异种移植,因猪易于饲养繁殖,器官尺寸与人类相匹配,且生理和代谢方面与人类极为相近,人猪共患病发生的可能性较小,涉及伦理道德争议少,并可通过人为的基因修饰来增强供体组织器官的匹配性。因此,猪被公认是最适合用作胰岛细胞及皮肤组织异种移植供体的物种。经过科学家们近半个多世纪的努力和探索,目前已筛选到近30个能有效缓解免疫学不相容性和克服功能性障碍的相关修饰基因,多种基因修饰的供体猪也被开发并进行了猪到非人灵长类的异种移植试验。
尽管猪作为人类器官移植的来源得到了认可,但异种组织移植仍面临着跨物种间疾病感染的风险、免疫学不相容性和功能性障碍三大问题使得猪胰岛细胞及皮肤组织作为异种移植面临着严峻的挑战,还不足以向临床应用医学转化,使得异种器官移植长期止步不前。通过此发明的应用,预采用目前先进的CRISPR/Cas9基因编辑技术,将一些容易引发移植免疫排斥反应的相关基因敲除,并加入一些人源化的相关修饰基因,将猪的组织器官进行人源化基因修饰,获得可供异种组织器官移植的抗免疫排斥反应以及人源化基因修饰猪,这在一定程度上就为解决糖尿病患者胰岛细胞移植及一些部队官兵受损或衰竭的皮肤等组织移植做准备,为挽救糖尿病患者的生命及部队烧伤士兵的皮肤等组织修复提供了异种移植的可能,具有重要的临床医学应用价值和研究意义。
发明内容
针对以上问题,本发明提供了一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,本方法只需在前期已构建的GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因修饰基础供体猪的基础上,再敲除B2M两个基因,定点插入hCD47、A20、CTLA4-Ig三个修饰基因,尽可能地提高胰岛细胞及皮肤异种移植的免疫耐受,降低免疫排斥、凝血功能障碍及炎症反应,保护异种移植的胰岛细胞、皮肤等组织移植物,对提高猪到非灵长类异种胰岛细胞及皮肤等组织异种移植的成功率具有重要的意义。
为达到上述目的,本发明提供如下技术方案:
一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,具体步骤为:
1)敲除引起抗原抗体免疫排斥反应的三个主要抗原基因
在上述猪成纤维细胞系的基础上,利用CRISPR/Cas9基因编辑技术敲除GGTA1、CMAH、B4GalNT2三个能引起抗原抗体免疫排斥反应的抗原基因;
2)定点插入hCD55、hCD59、hCD46三个人源化修饰基因
在上述GTKO/CMAHKO/B4GalNT2KO三基因修饰的基础上,再定点插入hCD55、hCD59、hCD46三个人源化修饰基因,获得GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因修饰的猪成纤维细胞系;
3)体细胞核移植和胚胎移植
利用体细胞核移植技术,将筛选获得的GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因细胞系进行体细胞核移植和胚胎移植,待妊娠时取出胎儿鉴定胎儿基因的编辑情况,分离培养获得六基因修饰的异种移植基础供体猪成纤维细胞系;
4)在上述基础猪胎儿成纤维细胞系上再敲除B2M基因,定点插入hCD47、A20、CTLA4-Ig四个修饰基因,经细胞筛选、体细胞核移植和胚胎移植后获得多基因修饰的胰岛细胞及皮肤等组织异种器官移植供体猪;
5)对上述所获得的多基因修饰的胰岛细胞及皮肤等组织异种移植供体猪进行功能验证,获得适合胰岛细胞及皮肤等组织异种移植的供体猪。
步骤4)中,在异种器官移植基础供体猪的基础上,针对异种胰岛细胞及皮肤等组织移植面临的免疫排斥和凝血功能障碍,采用基因定点插入和敲除策略,再将调控异种胰岛细胞及皮肤等组织移植引起的免疫排斥、免疫耐受、凝血功能障碍及炎症反应相关基因B2M、hCD47、A20、CTLA4-Ig进行修饰,获得异种胰岛细胞及皮肤等组织移植的供体猪。
步骤5)中,对多基因修饰的胰岛细胞及皮肤等组织异种移植供体猪所进行的功能验证主要包括人血清介导的细胞毒性实验、人补体介导的细胞毒性实验、补体激活实验、抗原抗体结合分析、细胞免疫组化分析、凝血相关检测和淋巴细胞活性检测等。
本发明公开了一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,在初步解决异种移植生物安全、免疫学不相容性及功能性障碍三大共性问题的基础上,该方法采用适合的针对胰岛细胞及皮肤等组织异种移植基因组合修饰策略,在GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因修饰基础供体猪的基础上,再敲除B2M基因,定点插入hCD47、A20、CTLA4-Ig三个修饰基因,最大限度地降低异种组织移植免疫排斥反应及凝血功能性障碍,这对开发真正有效的胰岛细胞及皮肤等组织异种移植供体猪具有重要的意义。
附图说明
图1是本发明的流程图。
具体实施方式
为了使本发明的目的、技术方案和有益效果更加清楚,下面将对本发明的优选实施例进行详细的说明,以方便技术人员理解。
实施例1
一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,具体操作过程按以下步骤进行:
1)敲除引起抗原抗体免疫排斥反应的三个主要抗原基因
在猪成纤维细胞系的基础上,利用CRISPR/Cas9基因编辑技术敲除GGTA1、CMAH、B4GalNT2三个能引起抗原抗体免疫排斥反应的抗原基因;
2)定点插入hCD55、hCD59、hCD46三个人源化修饰基因
在上述GTKO/CMAHKO/B4GalNT2KO三基因修饰的基础上,再定点插入hCD55、hCD59、hCD46三个人源化修饰基因,获得GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因修饰的猪成纤维细胞系;
3)体细胞核移植和胚胎移植
利用体细胞核移植技术,将筛选获得的GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因细胞系进行体细胞核移植和胚胎移植,待妊娠时取出胎儿鉴定胎儿基因的编辑情况,分离培养获得六基因修饰的异种移植基础供体猪胎儿成纤维细胞系;
4)在上述基础猪胎儿成纤维细胞系上再敲除B2M基因,定点插入hCD47、A20、CTLA4-Ig四个修饰基因,经细胞筛选、体细胞核移植和胚胎移植后获得多基因修饰的胰岛细胞及皮肤等组织异种器官移植供体猪。
实施例2
一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,具体操作过程按以下步骤进行:
一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,具体步骤为:
1)敲除引起抗原抗体免疫排斥反应的三个主要抗原基因
在猪成纤维细胞系的基础上,利用CRISPR/Cas9基因编辑技术敲除GGTA1、CMAH、B4GalNT2三个能引起抗原抗体免疫排斥反应的抗原基因;
2)定点插入hCD55、hCD59、hCD46三个人源化修饰基因
在上述GTKO/CMAHKO/B4GalNT2KO三基因修饰的基础上,再定点插入hCD55、hCD59、hCD46三个人源化修饰基因,获得GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因修饰的猪成纤维细胞系;
3)体细胞核移植和胚胎移植
利用体细胞核移植技术,将筛选获得的GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因细胞系进行体细胞核移植和胚胎移植,待妊娠30天时取出胎儿鉴定胎儿基因的编辑情况,分离培养获得七基因修饰的异种移植基础供体猪成纤维细胞系;
4)在上述基础猪胎儿成纤维细胞系上再敲除B2M基因,定点插入hCD47、A20、CTLA4-Ig四个修饰基因,经细胞筛选、体细胞核移植和胚胎移植后获得多基因修饰的胰岛细胞及皮肤等组织异种器官移植供体猪;
5)对上述所获得的多基因修饰的胰岛细胞及皮肤等组织异种移植供体猪进行功能验证,获得适合胰岛细胞及皮肤等组织异种移植的供体猪。
步骤4)中,在异种器官移植基础供体猪的基础上,针对异种胰岛细胞及皮肤等组织移植面临的免疫排斥和凝血功能障碍,采用基因定点插入和敲除策略,再将调控异种胰岛细胞及皮肤等组织移植引起的免疫排斥、免疫耐受、凝血功能障碍及炎症反应相关基因B2M、hCD47、A20、CTLA4-Ig进行特异修饰,获得异种胰岛细胞及皮肤等组织移植的供体猪。
步骤5)中,对多基因修饰的胰岛细胞及皮肤等组织异种移植供体猪所进行的功能验证主要包括人血清介导的细胞毒性实验、人补体介导的细胞毒性实验、补体激活实验、抗原抗体结合分析、细胞免疫组化分析、凝血相关检测和淋巴细胞活性检测等。
利用实施例中构建的一种适合胰岛细胞及皮肤等组织异种移植的供体猪,在初步解决了异种移植生物安全性、抗原免疫排斥及器官功能性障碍等问题的基础猪供体上,再将调控异种胰岛细胞及皮肤等组织移植引起的免疫排斥、免疫耐受、凝血功能障碍及炎症反应相关基因B2M、hCD47、A20、CTLA4-Ig进行特异表达修饰,获得多基因共同修饰的适合胰岛细胞及皮肤等组织异种移植的供体猪,该基因组合编辑策略科学可靠,使胰岛细胞及皮肤异种移植的成功率更高,将极大地促进胰岛细胞及皮肤等组织异种器官移植的研发进程,对利用基因组合修饰策略来开发真正有效的胰岛细胞及皮肤等组织异种移植供体猪具有重要的应用价值。
最后说明的是,以上优选实施例仅用于说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (2)
1.一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法,其特征在于:具体步骤为:
1)敲除引起抗原抗体免疫排斥反应的三个主要抗原基因
在猪胎儿成纤维细胞系的基础上,利用CRISPR/Cas9基因编辑技术敲除GGTA1、CMAH、B4GalNT2三个能引起抗原抗体免疫排斥反应的基因;
2)定点插入hCD55、hCD59、hCD46三个人源化修饰基因
在GTKO/CMAHKO/B4GalNT2KO三基因修饰的基础上,再定点插入hCD55、hCD59、hCD46三个人源化修饰基因,获得GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因修饰基础供体猪;
3)体细胞核移植和胚胎移植
利用体细胞核移植技术,将经筛选获得的GTKO/CMAHKO/B4GalNT2KO/hCD55/hCD59/hCD46六基因细胞系进行体细胞核移植,待妊娠时取出胎儿鉴定基因的编辑情况,分离培养获得六基因修饰基础供体猪胎儿成纤维细胞系;
4)在基础猪胎儿成纤维细胞的基础上,再敲除B2M基因,定点插入hCD47、A20、CTLA4-Ig三个修饰基因,利用体细胞核移植技术构建多基因修饰的胰岛细胞及皮肤等组织异种移植供体猪;
5)对步骤4)所获得的多基因修饰的胰岛细胞及皮肤等组织异种移植供体猪进行功能验证,获得适合胰岛细胞及皮肤等组织异种移植的供体猪。
2.根据权利要求1所述的一种适合胰岛细胞及皮肤组织异种移植供体猪的构建方法,其特征在于:步骤5)中,功能验证包括人血清介导的细胞毒性实验、人补体介导的细胞毒性实验、补体激活实验、抗原抗体结合分析、细胞免疫组化分析、凝血相关检测和淋巴细胞活性检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910497785.0A CN110373389A (zh) | 2019-06-10 | 2019-06-10 | 一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910497785.0A CN110373389A (zh) | 2019-06-10 | 2019-06-10 | 一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110373389A true CN110373389A (zh) | 2019-10-25 |
Family
ID=68250019
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910497785.0A Pending CN110373389A (zh) | 2019-06-10 | 2019-06-10 | 一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110373389A (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111778251A (zh) * | 2020-07-14 | 2020-10-16 | 金佩奇生物科技(南京)有限公司 | 敲除猪异种抗原的基因的gRNA及其应用 |
WO2021139722A1 (en) * | 2020-01-07 | 2021-07-15 | Hangzhou Qihan Biotechnology Co., Ltd. | Methods and compositions for production of xenogeneic islet cells and treatment of insulin-resistant or -deficient conditions with the same |
WO2022144856A1 (en) * | 2020-12-31 | 2022-07-07 | Crispr Therapeutics Ag | Universal donor cells |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108473963A (zh) * | 2015-09-09 | 2018-08-31 | 雷维维科公司 | 用于异种移植的多重转基因猪 |
-
2019
- 2019-06-10 CN CN201910497785.0A patent/CN110373389A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108473963A (zh) * | 2015-09-09 | 2018-08-31 | 雷维维科公司 | 用于异种移植的多重转基因猪 |
Non-Patent Citations (3)
Title |
---|
CHEN Y等: "Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59 and fucosyltransferase multiple transgenic donors", 《XENOTRANSPLANTATION》 * |
KONRAD FISCHER等: "Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing", 《SCIENTIFIC REPORTS》 * |
王唯予 等: "肾脏-胰岛联合移植研究进展", 《实用器官移植电子杂志》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021139722A1 (en) * | 2020-01-07 | 2021-07-15 | Hangzhou Qihan Biotechnology Co., Ltd. | Methods and compositions for production of xenogeneic islet cells and treatment of insulin-resistant or -deficient conditions with the same |
CN111778251A (zh) * | 2020-07-14 | 2020-10-16 | 金佩奇生物科技(南京)有限公司 | 敲除猪异种抗原的基因的gRNA及其应用 |
WO2022012512A1 (zh) * | 2020-07-14 | 2022-01-20 | 金佩奇生物科技(南京)有限公司 | 敲除猪异种抗原的基因的gRNA及其应用 |
WO2022144856A1 (en) * | 2020-12-31 | 2022-07-07 | Crispr Therapeutics Ag | Universal donor cells |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Bourret et al. | Human–animal chimeras: ethical issues about farming chimeric animals bearing human organs | |
CN110373389A (zh) | 一种适合胰岛细胞及皮肤等组织异种移植供体猪的构建方法 | |
Fujiwara et al. | Promoting roles of embryonic signals in embryo implantation and placentation in cooperation with endocrine and immune systems | |
CN110373391A (zh) | 一种适合心脏异种移植供体猪的构建方法 | |
Song et al. | In vitro spermatogenesis: A long journey to get tails | |
Billingham et al. | Further studies in tissue homotransplantation in cattle | |
Zeleniak et al. | De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids | |
CN109844093A (zh) | 通过降低t细胞区室中的自身反应性来治疗免疫病症相关疾病的方法 | |
CN110295143A (zh) | 有关胚胎移植延迟和使用外周血单核细胞的体外受精方法 | |
CN106701664A (zh) | 一种以睾丸支持细胞为滋养层提高精子活力的方法 | |
Gill-Peterson | IMPLANTING PLASTICITY INTO SEX AND TRANS/GENDER: animal and child metaphors in the history of endocrinology | |
Rossi et al. | Technologies for the production of fertilizable mammalian oocytes | |
Mesbah et al. | Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary | |
CN110423720A (zh) | 一种羊膜上皮干细胞诱导分化为功能性胰岛β细胞的方法及其应用 | |
Medvinsky et al. | Analysis and manipulation of hematopoietic progenitor and stem cells from murine embryonic tissues | |
RU2511395C2 (ru) | Предшественник искусственной почки и способ его получения | |
Lopata | History of the Egg in Embryology | |
CN110373392A (zh) | 一种适合肾脏异种移植供体猪的构建方法 | |
US20030149996A1 (en) | Method for producing polyclonal and monoclonal antibodies | |
Seifert et al. | A remarkable rodent: Regeneration and reproduction in spiny mice (Acomys) | |
Costa et al. | Distinct in vitro properties of embryonic and extraembryonic fibroblast-like cells are reflected in their in vivo behavior following grafting in the adult mouse brain | |
CN110373390A (zh) | 一种异种器官移植基础供体猪的构建方法 | |
CN102286427A (zh) | 用动物生产人的造血干细胞 | |
Depierreux et al. | Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry | |
CN107267445A (zh) | 一种提高哺乳动物体外受精‑胚胎移植着床成功率的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |