Targeted Disruption of the Pemphigus Vulgaris Antigen (Desmoglein 3) Gene in Mice Causes Loss of Keratinocyte Cell Adhesion with a Phenotype Similar to Pemphigus Vulgaris

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell–cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 −/− mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8–10 d weighed less than DSG3 +/− or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and “tombstoning” of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 −/− mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.     

Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.     

Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy

Immune-deficient Rag2(-/-) mice were used as nuclear donors for transfer into enucleated oocytes, and the resulting blastocysts were cultured to isolate an isogenic embryonic stem cell line. One of the mutated alleles in the Rag2(-/-) ES cells was repaired by homologous recombination, thereby restoring normal Rag2 gene structure. Mutant mice were treated with the repaired ES cells in two ways. (1) Immune-competent mice were generated from the repaired ES cells by tetraploid embryo complementation and were used as bone marrow donors for transplantation. (2) Hematopoietic precursors were derived by in vitro differentiation from the repaired ES cells and engrafted into mutant mice. Mature myeloid and lymphoid cells as well as immunoglobulins became detectable 3-4 weeks after transplantation. Our results establish a paradigm for the treatment of a genetic disorder by combining therapeutic cloning with gene therapy.     

Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle

Desmosomes are critical for the tissue integrity of stratified epithelia and their appendages. Desmogleins (DSGs) and desmocollins (DSCs) are transmembrane desmosomal cadherins that interact extracellularly to link neighboring epithelial cells. We recently identified a new member of the DSG family, designated desmoglein 4, whose mutations cause hypotrichosis in human, mouse and rat. In this study, we analyzed in detail the expression domains of human desmoglein 4 protein (DSG4) in human skin relative to differentiation markers and other DSGs. Our results show that DSG4 protein is expressed in the more highly differentiated layers of the epidermis. This expression pattern in vivo is recapitulated in highly differentiated HaCaT human keratinocytes and normal human keratinocytes in vitro. In the human hair follicle, DSG4 is expressed specifically in the hair shaft cortex, the lower hair cuticle, and the upper inner root sheath (IRS) cuticle. Using a green fluorescent protein-tagged version of mouse or rat desmoglein 4 protein (Dsg4) and immuno-electron microscopy, we demonstrate that Dsg4 localizes to desmosomes both in vitro and in vivo. The highly specific expression pattern of DSG4 in the human hair follicle, combined with the phenotype of rodent models and human patients with desmoglein 4 mutations, underscores the importance of this adhesion molecule in the integrity of the hair shaft.     

Establishment of a novel embryonic stem cell line by a modified procedure

To generate mutant mice, embryonic stem (ES) cells are used as a vehicle for introducing mutations. The establishment of ES cells is diffucult because it requires specific skills and it is time-consuming. We established a novel ES cell line derived from hybrid mice between C57BL/6 and DBA/2 using a modified method. To collect a large number of preimplantational embryos, we collected embryos at the 8-cell stage and cultured them to blastocysts, whereas the usual procedure of preparing the delayed blastocysts demands technical skills. To eliminate unnecessary female cells at an initial stage of inner cell mass culture, male clones were selected by polymerase chain reaction to detect the mouseSry gene. The established ES cell line efficiently contributed to the germ-line when injected into 8-cell embryos of ICR mice. This potency was maintained after manipulation throughout gene targeting.Abbreviations DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - FIAU 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil - LIF leukemia inhibitory factor - NEAA non-essential amino acids     

Nuclear transfer-mediated rescue of the nuclear genome of nonviable mouse cells frozen without cryoprotectant

Nuclear transfer (NT) provides an opportunity for clonal amplification of a nuclear genome of interest. Here, we report NT-mediated reprogramming with frozen mouse cells that were nonviable because they were frozen at -80 degrees C for up to 342 days without a cryoprotectant. We derived eight embryonic stem (ES) cell lines from cloned blastocysts by conventional NT procedure and five ntES (nuclear transfer embryonic stem) cell lines by a modified NT procedure in which a whole cell instead of a nucleus was injected into an enucleated oocyte. Chromosome analysis revealed that 12 of 13 ntES cell lines have normal karyotypes. On injection of ntES cells into tetraploid blastocysts to generate clonal mice that are nearly completely ntES-cell derived, live pups were obtained; four clonal mice survived until adulthood. On injection of ntES cells into diploid blastocysts, chimeric mice with a high somatic ES cell contribution were generated; germ-line transmission was obtained. Our findings indicate that chromosome stability and genomic integrity can be maintained in mouse somatic cells after freezing without cryoprotection and that NT and ES cell techniques can rescue the genome of these cells.     

Anti-desmoglein 3 (Dsg3) monoclonal antibodies deplete desmosomes of Dsg3 and differ in their Dsg3-depleting activities related to pathogenicity

Pemphigus vulgaris (PV) is an autoimmune blistering disease, characterized by the loss of cell-cell adhesion between epidermal keratinocytes and the presence of autoantibody against desmoglein 3 (Dsg3), which provides adhesive integrity to desmosomes between adjacent keratinocytes. We have previously shown that PV-IgG purified from patients depletes desmosomes of Dsg3. However, PV-IgG contains not only antibodies against a variety of different epitopes of Dsg3 but also against other unknown antigens. Therefore, we examined whether the Dsg3-depleting activity of PV-IgG is generated specifically by anti-Dsg3 activity in a human squamous cell carcinoma cell line (DJM-1) and normal human keratinocytes by using four different pathogenic and nonpathogenic monoclonal antibodies against Dsg3. We demonstrate that these monoclonal antibodies deplete cells and desmosomes of Dsg3, as PV-IgG does. Individual monoclonal anti-Dsg3 antibodies display characteristic limits to their Dsg3-depleting activity, which correlates with their pathogenic activities. In combination, these antibodies exert a cumulative or synergistic effect, which may explain the potent Dsg3-depleting capability of PV-IgG, which is polyclonal. Finally, although Dsg3-depletion activity correlated with AK-monoclonal antibody pathogenicity in mouse models, the residual level of Dsg3, when below approximately 50%, does not correlate with the adhesive strength index in the present study. This may suggest that although the Dsg3 depletion is not indicative for adhesive strength, the level of Dsg3 can be used as a read-out of pathogenic changes within the cell and that the Dsg3 depletion from desmosomes plays an important role in skin fragility or susceptibility to blister formation in PV patients.     

Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines.

Gene targeting in embryonic stem (ES) cells allows the production of mice with specified genetic mutations. Currently, germline-competent ES cell lines are available from only a limited number of mouse strains, and inappropriate ES cell/host blastocyst combinations often restrict the efficient production of gene-targeted mice. Here, we describe the derivation of C57BL/6J (B6) ES lines and compare the effectiveness of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyr(c)-2J (c2J), for the production of germline chimeras. We found that when B6 ES cells were injected into c2J host blastocysts, a high rate of coat-color chimerism was detected, and germline transmission could be obtained with few blastocyst injections. In all but one case, highly chimeric mice transmitted to 100% of their offspring. The injection of B6 ES cells into FVB blastocysts produced some chimeric mice. However; the proportion of coat-color chimerism was low, with many more blastocyst injections required to generate chimeras capable of germline transmission. Our data support the use of the coisogenic albino host strain, c2J, for the generation of germline-competent chimeric mice when using B6 ES cells.     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

Extracellularly truncated desmoglein 1 compromises desmosomes in MDCK cells

The formation and stability of epithelial tissue involves cell adhesion and the connection of the intermediate filaments of contiguous cells, mediated by desmosomes. The cadherin family members Desmocollins (Dsc) and Desmogleins (Dsg) mediate desmosome extracellular adhesion. The main intracellular molecules identified linking Dscs and Dsgs with the intermediate filament network are Plakoglobin (PG), Plakophilins (PPs) and Desmoplakin (DP). Previous studies on desmosome-mediated adhesion have focused on the intracellular domains of Dsc and Dsg because of their capacity to interact with PG, PPs and DP. This study examines the role of the extracellular domain of Dsg1 upon desmosome stability in MDCK cells. Dsg1 was constructed containing an extracellular deletion (Dsg delta 1EC) and was expressed in MDCK cells. A high expressor Dsg delta 1EC/MDCK clone was obtained and analysed for its capacity to form desmosomes in cell monolayers and when growing under mechanical stress in three-dimensional collagen cultures. Phenotypic changes associated with the ectopic expression of Dsg1 delta EC in MDCK cells were: disturbance of the cytokeratin network, a change in the quality and number of desmosomes and impairment of the formation of cysts in suspension cultures. Interestingly, Dsg1 delta EC was not localized in desmosomes, but was still able to maintain its intracytoplasmic interaction with PG, suggesting that the disruptive effects were largely due to PG and/or PP sequestration.     

Culturing in vitro produced blastocysts in sequential media promotes ES cell derivation

Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K(+)) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM-CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM-CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM-CBM versus 18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation.     

品系对小鼠胚胎干细胞分离效率的影响

为了充分利用小鼠胚胎干(ES)细胞,就必须从众多小鼠品系中分离ES细胞系。本研究通过传统的成纤维细胞饲养层法,从CD-1、129/Sv、C57BL/6J和129/Sv×C57BL/6J四种不同遗传背景的小鼠中分离得到12个ES细胞系,而从KM小鼠没有得到ES细胞系。所有的ES细胞系都具有典型的ES细胞特征,AKP染色呈阳性。从四种不同遗传背景的ES细胞系得到了包含多种组织的畸胎瘤;与桑椹胚聚合后,都得到了生殖系嵌合体。结果表明:品系对小鼠ES细胞的分离有显著影响,利用129小鼠以及包含129小鼠遗传背景的杂交小鼠都较容易分离ES细胞,由ES细胞得到生殖系嵌合体的效率在不同品系间有显著差异,从杂交ES细胞比近交ES细胞中更容易得到生殖系嵌合体。     

The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro.

Previous studies have suggested that the vav protooncogene plays an important role in hematopoiesis. To study this further, we have ablated the vav protooncogene by homologous recombination in embryonic stem (ES) cells. Homozygous vav (-/-) ES clones differentiate normally in culture and generate cells of erythroid, myeloid and mast cell lineages. Mice heterozygous for the targeted vav allele do not display any obvious abnormalities. However, homozygous embryos die very early during development. Crosses of vav (+/-) heterozygous mice yield apparently normal vav (-/-) E3.5 embryos but not post-implantation embryos (> or = E7.5). Furthermore, homozygous vav (-/-) blastocysts do not hatch in vitro. These results indicate that vav is essential for an early developmental step(s) that precedes the onset of hematopoiesis. Consistent with the phenotypic analysis of vav (-/-) embryos, we have identified Vav immunoreactivity in the extra-embryonic trophoblastic cell layer but not in the inner embryonic cell mass of E3.5 preimplantation embryos or in the egg cylinder of E6.5 and E7.5 post-implantation embryos. These results suggest that the vav gene is essential for normal trophoblast development and for implantation of the developing embryo.     

At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation

Chimeric or entirely embryonic stem (ES) cell-derived mice ("ES mice") can be produced by injecting ES cells into diploid (2n) or tetraploid (4n) host blastocysts, respectively. Usually, between 10 and 15 ES cells are injected into the host blastocyst, but it is not clear how many of the injected cells contribute to the somatic lineages, thus serve as "founder cells" of the embryo proper. We have used genetically labeled ES cells to retrospectively determine the number of founder ES cells that generate the somatic lineages of chimeric and of ES mice. ES cell clones individually labeled with provirus were mixed in equal numbers and injected into 2n or 4n blastocysts to generate chimeric or ES mice. Southern analysis of DNA from the resulting animals indicated that the somatic lineages were most often derived from one or two and sometimes from up to three founder ES cells. The number of founder cells was independent of the total number of cells injected into the host blastocysts. Our results are consistent with the notion that constraints of the host embryo restrict the number of ES cells that can contribute to a chimeric or an ES mouse.     

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.     

Comparison of tetraploid blastocyst microinjection of outbred Crl:CD1(ICR), hybrid B6D2F1/Tac, and inbred C57BL/6NTac embryos for generation of mice derived from embryonic stem cells

Embryo electrofusion and tetraploid blastocyst microinjection is a modification of the traditional embryonic stem cell (ES cell)-based method to generate targeted mutant mice. Viability of tetraploid embryos is reportedly lower than with diploid embryos, with considerable interstrain variation. Here we assessed fetus and pup viability after ES cell microinjection of tetraploid blastocysts derived from outbred, hybrid, and inbred mice. Two-cell mouse embryos (C57BL/6NTac [B6], n = 788; B6D2F1/Tac [BDF1], n = 1871; Crl:CD1(ICR) [CD1], n = 1308) were electrofused; most resultant tetraploid blastocysts were injected with ES cells and surgically transferred into pseudopregnant recipient mice. Reproductive tracts were examined at midgestation for embryologic studies using B6 and BDF1 blastocysts; implantation sites and viable fetuses were counted. Pregnancies were carried to term for studies of targeted mutant mice using BDF1 and CD1 blastocysts, and pup yield was evaluated. Electrofusion rates of 2-cell embryos did not differ among B6, BDF1, and CD1 mice (overall mean, 92.8% +/- 5.4%). For embryologic studies, 244 B6 blastocysts were surgically transferred and 1 fetus was viable (0.41%), compared with 644 BDF1 blastocysts surgically transferred and 88 viable fetuses (13.7%). For targeted mutant mouse studies, 259 BDF1 blastocysts were surgically transferred yielding 10 pups (3.9%); 569 CD1 blastocysts yielded 44 pups (7.7%).     

Isolation and characterization of embryonic stem cell-like cells from in vitro produced goat (Capra hircus) embryos

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P?相似文献   

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.     

Establishment of a germ-line competent C57BL/6 embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.