55 results about "Nuclear transplantation" patented technology

The application discloses an embryo culture method and embryo culture solution for improving the quality of pig somatic cell nuclear transfer embryos, wherein a small molecule substance X1 capable of specifically combining with the RepA domain of Xist is added into a pig early embryo culture solution, so as to improve the development quality of pig SCNT embryos.

The application discloses a gene editing system for constructing a three-gene (GHR gene, IGF1 gene and IGF2 gene) combined mutation small pig nuclear transfer donor cell and application thereof. The application provides a kit comprising GHR-E5-gRNA2 shown in SEQ ID NO: 36, GHR-E5-gRNA3 shown in SEQ ID NO: 37, IGF1-E4-gRNA1 shown in SEQ ID NO: 38, IGF1-E4-gRNA2 shown in SEQ ID NO: 39, IGF2-E4-gRNA2 shown in SEQ ID NO: 40, IGF2-E4-gRNA7 shown in SEQ ID NO: 41 and NCN protein. The NCN protein is a Cas9 protein or a fusion protein with the Cas9 protein. The application adopts CRISPR / Cas9 technology combined with double gRNA editing to perform combined knockout of the GHR gene, the IGF1 gene and the IGF2 gene, and obtains a single cell clone with three-gene combined knockout, thereby laying a foundation for breeding small pigs and growth and development disorder or retardation model pigs through somatic cell nuclear transfer animal cloning technology in the later stage.

The invention relates to the technical field of animal breeding, in particular to a method for breeding black sheep. The method comprises the following steps: introducing an expression cassette into a cell to obtain a transgenic cell; and carrying out somatic cell nuclear transplantation cloning by adopting the transgenic cells to obtain the black sheep. The expression cassette comprises an intron of PK5 and beta-globin, and an EDN3 gene, the PK5 comprises a nucleotide sequence as shown in SEQ ID NO. 2; and the EDN3 gene comprises a nucleotide sequence as shown in SEQ ID NO. 4. A specific expression cassette and a carrier are researched, the black sheep of which the skin, hair, tongue, oral mucosa, eye white mucosa, epididymis and ears all become black can be prepared through somatic cell cloning on the basis of cells edited by the carrier, and important application value and economic value are achieved.

The application discloses a gene editing system for constructing a muscle weakness disease model pig nuclear transfer donor cell with a DOK7 gene mutation and application thereof. The application provides application of DOK7-gRNA2 shown in SEQ ID NO: 16, DOK7-gRNA4 shown in SEQ ID NO: 17 and NCN protein in preparation of a kit. The application further provides a method for preparing a recombinant cell, comprising the following steps: co-transfecting a pig cell with DOK7-gRNA2, DOK7-gRNA4 and NCN protein to obtain a recombinant cell. The recombinant cell is a recombinant cell with a mutated DOK7 gene. The use of any of the above kits is to prepare a recombinant cell, to prepare a muscle weakness model pig, to prepare a muscle weakness cell model, a muscle weakness tissue model or a muscle weakness organ model. The application has great application value for research and development of muscle weakness disease drugs and revealing the pathogenesis of the disease.

This invention discloses a gene editing system for constructing a porcine nuclear transplantation donor cell model of atherosclerosis with double gene mutations in AF and its applications. The invention provides a kit comprising plasmid pKG-U6gRNA (APOE-E2-gRNA2), a target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 25 (FBN1-gRNA4), a target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 26 (FBN1-gRNA6), and FBN1-mutant-ss163 as shown in SEQ ID NO: 27. The kit is used for: preparing recombinant cells; preparing porcine atherosclerosis models; preparing atherosclerosis cell models, atherosclerosis tissue models, or atherosclerosis organ models. The plasmid pKG-U6gRNA (APOE-E2-gRNA2) is transcribed to obtain sgRNA with a target sequence binding region as shown in nucleotides 1-20 of SEQ ID NO: 11. APOE‑E2‑gRNA2 This invention lays the foundation for developing pig models of atherosclerosis through somatic cell nuclear transfer animal cloning technology.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for cataract disease models with MIP gene mutations and its applications. This invention provides the application of MIP-gRNA2 (SEQ ID NO: 18), MIP-gRNA3 (SEQ ID NO: 19), and NCN protein in a preparation kit. This invention also provides a method for preparing recombinant cells, comprising the following steps: co-transfecting porcine cells with MIP-gRNA2, MIP-gRNA3, and NCN protein to obtain recombinant cells. The recombinant cells are recombinant cells with a mutated MIP gene. The kits described above are used for: preparing recombinant cells; preparing porcine cataract models; preparing cataract cell models, cataract tissue models, or cataract organ models. This invention has significant application value for the development of cataract drugs and for elucidating the pathogenesis of this disease.

This invention discloses a method for constructing an immune animal model for preparing biofusion enzyme antibodies and its application. The method includes the following steps: designing and screening sgRNAs with high cleavage efficiency based on signal protein genes, and constructing an sgRNA-Cas9 expression vector; linearizing the plasmid by double enzyme digestion, and then ligating it with a signal protein gene fragment containing left and right homologous arms to obtain the Donor plasmid; co-transfecting the sgRNA-Cas9 expression vector and the Donor plasmid into target animal somatic cells, and screening to obtain positive somatic cells that stably integrate the target gene; using the positive somatic cells as nuclear donors for nuclear transfer to construct recombinant embryos, and transferring the recombinant embryos into recipient female animals; after delivery, identifying transgenic animal individuals carrying biofusion enzyme antibodies by genomic PCR. Based on this transgenic animal model, different target antibodies with clinical value can be developed.

The application discloses a kit for constructing an ATM gene mutation ataxia-telangiectasia model pig nuclear transfer donor cell. The application provides a kit comprising ATM-gRNA1 shown in SEQ ID NO: 16, ATM-gRNA4 shown in SEQ ID NO: 17 and NCN protein. The application also provides a method for preparing a recombinant cell: co-transfecting a pig cell with ATM-gRNA1, ATM-gRNA4 and NCN protein to obtain a recombinant cell. The recombinant cell is a recombinant cell with ATM gene mutation. The kit is used for: preparing a recombinant cell; preparing an ataxia-telangiectasia model pig; preparing an ataxia-telangiectasia cell model or an ataxia-telangiectasia tissue model or an ataxia-telangiectasia organ model. The application has great application value for research and development of ataxia-telangiectasia drugs and revealing the pathogenesis of the disease.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for ALS models with SOD1 gene mutations and its applications. This invention also provides a method for preparing recombinant cells: SOD1- g RNA1 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 18), SOD1- g RNA6 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 19), SOD1-mutant-ss160 (shown in SEQ ID NO: 20), and NCN protein (Cas9 protein or a fusion protein containing Cas9 protein) were co-transfected into porcine cells to obtain recombinant cells. This invention utilizes CRISPR / Cas9 technology combined with ssODN homologous recombination technology to perform point mutation gene editing of the SOD1 gene, mimicking the natural pathogenesis and genetic characteristics of ALS, and obtaining single-cell clones with precise point mutations in the SOD1 gene. This lays the foundation for later breeding of ALS disease model pigs using somatic cell nuclear transfer animal cloning technology.

The invention belongs to the technical field of nucleus visual identification, and discloses a method for identifying and predicting the nucleus position of an oocyte by using a computer image, which comprises the following steps of: dividing the oocyte into a mature oocyte and an immature oocyte, and acquiring an oocyte image; dividing the oocyte image into a mature oocyte image and an immature oocyte image; carrying out visual imaging operation on the nucleus of the oocyte, and meanwhile, carrying out image annotation on an oocyte photo; performing data amplification on images containing mature oocytes and cell nucleus positions, and dividing the images into a training set and a verification set; a computer vision framework is trained using the data set. According to the method disclosed by the invention, the cell nucleus position of the mammal oocyte is predicted by utilizing a computer visual identification technology, the enucleation accuracy is improved from 53.84% to 92.30%, and the accuracy of judging the cell nucleus position of the oocyte in a somatic cell nucleus transplantation process is improved.

This invention relates to the fields of gene editing and animal genetic breeding technology, and discloses a precise editing method for key genes regulating lipid metabolism in cattle and sheep. The method includes: constructing a comprehensive lipid metabolism network map integrating transcriptomic, proteomic, and metabolomic data; using a graph neural network to identify key node genes with high flow weights and their negative feedback pathways; simulating cascade reactions after gene perturbation to assess compensatory effects; screening for multi-gene combinations that do not induce significant compensation and can synergistically regulate fat deposition and muscle growth; designing expression vectors containing multiple guide RNA sequences to achieve simultaneous and precise editing; and obtaining edited individuals and validating the phenotype through embryonic or somatic cell nuclear transfer. This invention, through system-level multi-target synergistic editing, avoids metabolic imbalances, improves editing effectiveness, and promotes muscle development while reducing fat deposition, providing a safe and efficient technical path for precision breeding of cattle and sheep.

This invention discloses a method and its application for constructing a sickle cell anemia model pig with HBB gene mutations using nuclear transfer donor cells. This invention provides the application of HBB-gU1, HBB-gD3, HBB-mutant-ss174, and NCN proteins in a preparation kit. The kit is used for: preparing recombinant cells; preparing sickle cell anemia model pigs; preparing sickle cell anemia cell models, sickle cell anemia tissue models, or sickle cell anemia organ models. HBB-gU1 is an sgRNA, and its target sequence binding region is shown as nucleotides 3-22 of SEQ ID NO: 18. HBB-gD3 is an sgRNA, and its target sequence binding region is shown as nucleotides 3-22 of SEQ ID NO: 19. HBB-mutant-ss174 is a single-stranded DNA molecule shown in SEQ ID NO: 20. The NCN protein is a Cas9 protein or a fusion protein containing a Cas9 protein. This invention has significant application value for the development of drugs for sickle cell anemia and for elucidating the pathogenesis of the disease.

The application discloses a gene editing system for constructing a GP130 gene mutation gastric cancer model pig nuclear transfer donor cell and application thereof. The gene editing system comprises a high-efficiency Cas9 protein prepared according to the method of the application, a high-efficiency target gRNA for the GP130 gene screened, and a single-chain Donor DNA containing a GP130 mutation site, and the optimal use amount ratio of the components of the system is optimized, and finally the single-cell clone ratio of the target site point mutation is 22.5%, which is much higher than the conventional point mutation efficiency (<5%).

The application discloses a CRISPR / cas system and application thereof in constructing INHA mutant high-fertility cloned pig nuclear donor cells. A CRISPR / Cas9 system for editing a pig INHA gene comprises a Cas9 expression vector and a gRNA expression vector; the Cas9 expression vector is a pKG-GE3 vector with a plasmid full sequence as shown in SEQ ID NO. 2; and the gRNA expression vector expresses gRNA with an expression sequence as shown in SEQ ID NO. 31. The gRNA combined with the Cas9 high-efficiency expression vector reformed by the application is used for gene editing, and the editing efficiency is significantly improved compared with that of the original vector.

This invention discloses a CRISPR system for constructing pig nuclear transfer donor cells for cataract models with YAP1 gene mutations and its applications. The invention provides the application of YAP1-gRNA2 (SEQ ID NO: 18), YAP1-gRNA3 (SEQ ID NO: 19), and NCN protein in a preparation kit; the kit is used for: preparing recombinant cells; preparing pig cataract models; preparing cataract cell models, cataract tissue models, or cataract organ models. This invention uses CRISPR / Cas9 technology combined with dual gRNA editing to knock out the YAP1 gene, simulating the natural pathogenesis and genetic characteristics of cataracts, and obtaining single-cell clones with the YAP1 gene knockout, laying the foundation for later breeding pig cataract models through somatic cell nuclear transfer animal cloning technology. This invention has significant application value for the development of cataract drugs and elucidating the pathogenesis of this disease.

This invention discloses a gene editing system for constructing porcine nuclear transfer donor cells for a vWF gene-mutated von Willebrand disease (VHD) model and its applications. The invention provides the application of gRNA-vWF-gU2 (SEQ ID NO: 15), gRNA-vWF-gD1 (SEQ ID NO: 16), and NCN protein in a preparation kit. The NCN protein is a Cas9 protein or a fusion protein containing Cas9. The kit is used for: preparing recombinant cells; preparing porcine VHD models; preparing VHD cell models, VHD tissue models, or VHD organ models. This invention also provides a method for preparing recombinant cells, comprising the following steps: co-transfecting porcine cells with gRNA-vWF-gU2, gRNA-vWF-gD1, and NCN protein to obtain recombinant cells. This invention has significant application value for the development of drugs for VHD and for elucidating the pathogenesis of this disease.

The present application relates to the field of embryo engineering, and particularly relates to application of mangiferin in promoting maturation of animal oocytes. The present application discloses that mangiferin can promote maturation of animal oocytes, especially maturation of pig oocytes; can improve in-vitro maturation efficiency of oocytes; can reduce the apoptosis rate of oocytes and improve the subsequent development potential of oocytes. Mangiferin can be used to promote in-vitro maturation of oocytes, and can be used to prepare or directly serve as a drug for improving in-vitro maturation and development of oocytes, thereby opening up a new application direction for mangiferin. The present application uses mangiferin to improve in-vitro maturation efficiency of oocytes, solves the problems of low maturation efficiency and long culture period, and provides more high-quality oocytes for somatic cell nuclear transfer, in-vitro fertilization and transgenic cloning by using the technical scheme of mangiferin in the present application, thereby having a wide application prospect in actual production.

The application discloses a method for constructing a MRAP2 gene mutation severe early-onset obesity model pig nuclear transfer donor cell. The application provides a kit, which comprises MRAP2-gRNA2 shown in SEQ ID NO: 16, MRAP2-gRNA3 shown in SEQ ID NO: 17, MRAP2-mutant-ss130 shown in SEQ ID NO: 18 and a fusion protein with Cas9 protein. The application also provides a method for preparing a recombinant cell: MRAP2-gRNA2, MRAP2-gRNA3, MRAP2-mutant-ss130 and NCN protein are co-transfected into a pig cell, so that a DNA molecule shown in SEQ ID NO: 18 is used to replace a DNA molecule shown in SEQ ID NO: 19 in chromosomal DNA of the pig cell. The application has great application value for research and development of an obesity treatment drug and revealing an onset mechanism of the disease.

The invention discloses a marker for evaluating the development potential of a pig somatic cell nuclear transfer embryo and application, and belongs to the technical field of development of the pig somatic cell nuclear transfer embryo. According to the invention, the endogenous retrovirus MLT2B2 is determined to be used as a functional regulator and a biomarker of the porcine embryo ZGA. The expression of the MLT2B2 in a 4-cell-stage SCNT embryo has obvious heterogeneity, and the embryo of the MLT2B2-generally shows ZGA retardation and low developmental potential. Single-embryo multi-omics analysis and pedigree tracking prove that the activation of the MLT2B2 in a 4-cell stage can be used as a predictive factor of the developmental ability of the SCNT embryo, that is, the MLT2B2 + embryo has recovered chromatin accessibility and transcription fidelity.

The present invention discloses a gene editing system for constructing a pig nuclear transplant donor cell for an OCA-1A albinism model and its application. The present invention provides a method for preparing a recombinant cell: the DNA molecule shown in SEQ ID NO: 19 in the chromosomal DNA of a pig cell is replaced with the DNA molecule shown in SEQ ID NO: 18 to obtain a recombinant cell. Implementation method: TYR-gRNA1, whose target sequence binding region is shown in nucleotides 3-22 of SEQ ID NO: 16, TYR-gRNA2, whose target sequence binding region is shown in nucleotides 3-22 of SEQ ID NO: 17, TYR-E1mut-ss126 shown in SEQ ID NO: 18, and NCN protein are co-transfected into pig cells. The present invention has significant application value for the research and development of drugs for OCA-1A albinism and for revealing the pathogenesis of the disease.

The application discloses a gene editing system for constructing an epigenetic disorder model pig nuclear transfer donor cell with a SETDB1 gene mutation and application thereof. The application provides application of SETDB1-gRNA1 shown in SEQ ID NO: 16, SETDB1-gRNA4 shown in SEQ ID NO: 17 and NCN protein in preparation of a kit. The application also provides a method for preparing a recombinant cell: SETDB1-gRNA1, SETDB1-gRNA4 and NCN protein are co-transfected into a pig cell to obtain the recombinant cell. The recombinant cell is a recombinant cell with a SETDB1 gene mutation. The kit is used for: preparing the recombinant cell; preparing an epigenetic disorder model pig; preparing an epigenetic disorder cell model or an epigenetic disorder tissue model or an epigenetic disorder organ model. The application has great application value for research and development of drugs for epigenetic disorder related diseases and revealing pathogenesis of the diseases.

The present invention discloses a gene editing system for constructing HNF1A gene mutation-producing MODY model pig nuclear transfer donor cells and its application. The present invention provides a method for preparing recombinant cells: the DNA molecule represented by SEQ ID NO: 18 is substituted for the DNA molecule represented by SEQ ID NO: 19 in the chromosomal DNA of a pig cell to obtain a recombinant cell. Specifically, the target sequence binding region HNF1A-gU2 represented by nucleotides 3-22 of SEQ ID NO: 16, the target sequence binding region HNF1A-gD1 represented by nucleotides 3-22 of SEQ ID NO: 17, the HNF1A-mutant-ss163 represented by SEQ ID NO: 18, and the NCN protein are co-transfected into pig cells. The recombinant cells are used as nuclear transfer donor cells for somatic cell cloning to obtain cloned pigs, namely, MODY type 3 diabetes model pigs. The present invention has significant application value in the development of drugs for MODY type 3 diabetes and in revealing the pathogenesis of the disease.

The present invention relates to a somatic cell nuclear transfer method and its application. This method uses a haploid embryonic stem cell system to obtain donor somatic cells in which one or more of 26 imprinted genes related to H3K27me3 are knocked out monoallelically. The results show that monoallelic knockout of one or more of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (such as one or more of the four genes Sfmbt2, Jade1, Gab1, and Smoc1) with H3K27me3 makes the imprinted expression patterns of these genes tend to be normal. At the same time, cloning using these donor somatic cells can increase the somatic cell cloning efficiency to 14%, while the somatic cell cloning efficiency of the wild-type control group is 0. In addition, for the cloned animals obtained by this method, the phenomenon of large placenta - large fetus is corrected.

This invention provides a method for preparing goats overexpressing the FOXL2 gene, belonging to the field of genetic engineering and transgenic animal technology. The method includes the following steps: transfecting intersex goat embryonic fibroblasts with a vector plasmid overexpressing the FOXL2 gene to obtain a stable monoclonal cell line overexpressing the FOXL2 gene; using the monoclonal cell line as a nuclear donor for nuclear transfer cloning in a recipient goat to obtain cloned goat embryos overexpressing the FOXL2 gene; and transferring the cloned goat embryos into a surrogate goat to produce goats overexpressing the FOXL2 gene. The nucleotide sequence of the FOXL2 gene is shown in SEQ ID NO.1. By overexpressing the FOXL2 gene in intersex goats, the regulatory function of the FOXL2 gene on horn traits is explored, providing insights for reducing or even overcoming the harm of intersex hornless syndrome and obtaining normal hornless dairy goat germplasm.

The invention discloses a method for improving the developmental capacity of a pig somatic cell nuclear transfer embryo and application, and belongs to the technical field of embryo culture. The invention aims to improve the success rate of pig somatic cell nucleus embryo transfer. The invention provides a method for improving the ex-embryonic developmental capacity of pig somatic cell nuclear transfer, which comprises the following steps: adding a small molecule inhibitor into a somatic cell culture solution used for somatic cell nuclear transfer to reduce somatic cell H3K9me3 modification, and carrying out somatic cell nuclear transfer by using PEF with reduced H3K9me3 modification as donor cells to improve the somatic cell nuclear transfer developmental capacity. The cell culture solution is prepared from the following components: a DMEM (Dulbecco Modified Eagle Medium) culture medium is used as a basic culture solution, and 15% fetal calf serum, OTS186935, F5446 and SETDB1-TTD-IN-1 are added into the basic culture medium. The culture solution can reduce H3K9me3 modification of donor cell PEF and improve the in-vitro developmental capacity of pig somatic cell nuclear transfer embryos.

This invention discloses a gene editing system for constructing TMPRSS6 gene-mutated porcine nuclear transfer donor cells for iron deficiency anemia and its applications. This invention provides the application of TMPRSS6-gRNA2 (SEQ ID NO: 16), TMPRSS6-gRNA4 (SEQ ID NO: 17), and NCN protein in a preparation kit. This invention also provides a method for preparing recombinant cells, comprising the following steps: co-transfecting porcine cells with TMPRSS6-gRNA2, TMPRSS6-gRNA4, and NCN protein to obtain recombinant cells. The recombinant cells are recombinant cells with a mutated TMPRSS6 gene. The kits described above are used for: preparing recombinant cells; preparing porcine models of iron deficiency anemia; preparing cell models, tissue models, or organ models of iron deficiency anemia. This invention has significant application value for the development of drugs for iron deficiency anemia and for elucidating the pathogenesis of this disease.

The application discloses application of a gene editing system in preparation of a spinal muscular atrophy model pig nuclear transfer donor cell with SMN1 gene mutation. The application provides application of SMN1-gRNA3 shown in SEQ ID NO: 16, SMN1-gRNA4 shown in SEQ ID NO: 17 and NCN protein in preparation of a kit. The application also provides a method for preparing a recombinant pig cell: SMN1-gRNA3, SMN1-gRNA4 and NCN protein are co-transfected into a pig cell to obtain a recombinant pig cell. The recombinant pig cell is a recombinant pig cell with SMN1 gene mutation. The kit is used for: preparing a recombinant pig cell; preparing a spinal muscular atrophy model pig; preparing a spinal muscular atrophy cell model or a spinal muscular atrophy tissue model or a spinal muscular atrophy organ model. The application has great application value for research and development of spinal muscular atrophy drugs and revealing the pathogenesis of the disease.

The invention belongs to the technical field of cytology, and discloses application of a chromatin remodeling factor Smarcb1 in preparation of a protein marker for regulating and controlling in-vitro cloned embryo epigenetic modification of animal cells. And the chromatin remodeling factor Smarcb1 comprises a variable spliceosome Smarcb1.1 of the chromatin remodeling factor Smarcb1. The invention relates to an oocyte transplanted by using a Smarcb1.1 expression vector constructed by the Smarcb1.1. According to the invention, it is determined that the Smarcb1 adjusts the development condition of the cloned embryo through cell nucleus reprogramming, and the action mechanism of the Smarcb1 on SCNT embryo development is disclosed, so that the developmental rate of the cloned embryo is improved, and the development and application of a somatic cell nucleus transfer technology are promoted.

The present invention provides methods and compostions to improve the efficiency of somatic cell nuclear transfer (SCNT) and the consequent production of nuclear transfer ESC (ntESC) and transgenic cells and / or non-human animals. More specifically, the present invention relates to the discovery that trimethylation of Histone H3-Lysine 9 (H3K9me3) in reprogramming resistant regions (RRRs) in the nuclear genetic material of donor somatic cells prevents efficient somatic cell nuclear reprogramming or SCNT. The present invention provide methods and compositions to decrease H3K9me3 in methods to improve efficacy of SCNT by exogenous or overexpression of the demethylase Kdm4 family and / or inhibiting methylation of H3K9me3 by inhibiting the histone methyltransferases Suv39h1 and / or Suv39h2.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for a metoprolol reductase deficiency model with SPR gene mutations and its applications. The invention provides a kit comprising SPR-gRNA1 (SEQ ID NO: 16), SPR-gRNA4 (SEQ ID NO: 17), and NCN protein. The invention also provides a method for preparing recombinant cells: co-transfecting porcine cells with SPR-gRNA1, SPR-gRNA4, and NCN protein to obtain recombinant cells. The recombinant cells are recombinant cells with a mutated SPR gene. The kit is used for: preparing recombinant cells; preparing porcine metoprolol reductase deficiency models; and preparing metoprolol reductase deficiency cell models, tissue models, or organ models. This invention has significant application value for the development of drugs for metoprolol reductase deficiency and for elucidating the pathogenesis of this disease.