Teratocarcinoma cells as vehicles for introducing specific mutant mitochondrial genes into mice

The immediate purpose of the experiment was to establish a means of introducing specific mitochondrially encoded mutant genes into mice. Mouse teratocarcinoma stem cells were used as vehicles for the cytoplasmic markers because of their known capacity for normal somatic and germinal differentiation after injection into blastocysts. The mutation of choice, chloramphenicol resistance (CAP(R)), was first produced in a melanoma cell line by mutagenesis and selection. The CAP(R) trait was then transferred from a resistant melanoma cell to a sensitive (CAP(S)) teratocarcinoma cell by fusing to the latter only the cytoplasmic portion of the CAP(R) donor. This indirect route demonstrated the cytoplasmic provenance of the mutation. Protein synthesis in mitochondria isolated from the cybrid, or cytoplasmic hybrid, cells was barely affected by chloramphenicol, in contrast to the inhibitory influence of the drug on mitochondria of the parent teratocarcinoma line. Cells of the cybrid clone resembled teratocarcinoma cells and retained their ability to form diverse tissues in solid tumors produced from subcutaneous grafts. Cells from the tumors were retransplanted and were tested periodically by culture in chloramphenicol; they were found to be stably CAP(R) even after 16 weeks in vivo in the absence of the selective agent. The CAP(R) cybrids were microinjected into blastocysts of another inbred strain and, after transfer to foster mothers, mosaic mice were obtained. They comprised both cybrid- and blastocyst-derived cells in various tissues, as indicated by strain-specific nuclear markers. These results demonstrate successful normal differentiation of the CAP(R) lineage in vivo. Teratocarcinoma cybrids thus offer a practical portal of entry of preselected mitochondrial genes into mice. This will ultimately permit in vivo investigation of maternally transmitted traits, of mitochondrial genetic influences in specialized cells, and of possible roles of cytoplasmic genes in clinical and disease states.     

Production of homoplasmic xenomitochondrial mice

The unique features of mtDNA, together with the lack of a wide range of mouse cell mtDNA mutants, have hampered the creation of mtDNA mutant mice. To overcome these barriers mitochondrial defects were created by introducing mitochondria from different mouse species into Mus musculus domesticus (Mm) mtDNA-less (rho(0)) L cells. Introduction of the closely related Mus spretus (Ms) or the more divergent Mus dunni (Md) mitochondria resulted in xenocybrids exhibiting grossly normal respiratory function, but mild metabolic deficiencies, with 2- and 2.5-fold increases in lactate production compared with controls. The transfer of this model from in vitro to in vivo studies was achieved by introducing Ms and Md mitochondria into rhodamine-6G-treated Mm mouse embryonic stem (ES) cells. The resultant xenocybrid ES cells remained pluripotent, and live-born chimerae were produced from both Ms and Md xenocybrid ES cells. Founder chimeric females (G(0)) were mated with successful germ-line transmission of Ms or Md mtDNA to homoplasmic G(1) offspring. These xenocybrid models represent the first viable transmitochondrial mice with homoplasmic replacement of endogenous mtDNA and confirm the feasibility of producing mitochondrial defects in mice by using a xenomitochondrial approach.     

Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.

Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell <==> tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.     

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G(1)- or G(2)/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.     

Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos

The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.     

Improved experimental procedures for achieving efficient germ line transmission of nonobese diabetic (NOD)-derived embryonic stem cells

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g., Idd locus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBS Lett., 455, 101-104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a "pure" NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.     

Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells.

We have constructed and characterized a polyoma virus-based plasmid that is maintained as an autonomously replicating extrachromosomal element (episome) in mouse embryonic stem (ES) cells. Plasmid pMGD20neo contains the polyoma origin of replication harboring a mutated enhancer (PyF101), a modified polyoma early region that encodes the large tumor (T) antigen only, and a gene that confers resistance to G418 (neo). After transfection, the plasmid replicates in ES cells and is maintained as an extrachromosomal element in 15% of G418-resistant clones. Integration of the plasmid DNA is undetectable for at least 28 cell generations. In one clone, the transfected DNA persists unaltered as an episome at 10-30 copies per cell for at least 74 cell generations in the presence of G418. Cells that maintain the autonomously replicating plasmid can efficiently replicate and maintain a second plasmid that carries the polyoma origin of replication. Independent vector-containing ES cell lines showed no significant alteration of the karyotype, and two cell lines yielded several chimeric animals when introduced into blastocysts, suggesting that the presence of an episomal element and expression of polyoma large T do not eliminate the ES cells' ability to populate an embryo. This system offers an efficient means for manipulating and analyzing various aspects of gene expression in ES cells.     

Cytoplasmic Inheritance of Chloramphenicol Resistance in Mouse Tissue Culture Cells

A chloramphenicol-resistant mutant, isolated from mouse A9 cells, was enucleated and fused with a nucleated chloramphenicol-sensitive mouse cell line. Resultant fusion products, cytoplasmic hybrids (or "cybrids"), were selected as resistant to chloramphenicol, and had the nuclear markers and chromosome complement of the chloramphenicol-sensitive parent. These cybrids appeared at the high frequency of 2-8 per 10(4) cells plated. Neither parent produced any colonies when plated under identical selective conditions. Fusion between enucleated chloramphenicol-sensitive cell fragments and the chloramphenicol-sensitive cell produced no resistant colonies, suggesting that chloramphenicol resistance is not due to an increase in the ratio of cytoplasm to nucleus. Furthermore, fusions between resistant and sensitive nucleated cells produced resistant hybrids at a frequency 100 times less than that of resistant cybrids. Thus, these stable chloramphenicol-resistant cybrids result from the fusion of a chloramphenicol-resistant cytoplasm with a chloramphenicol-sensitive cell. It is proposed, therefore, that chloramphenicol resistance is a cytoplasmically inherited characteristic in this mouse cell line.     

Hematopoiesis following disruption of the Pitx2 homeodomain gene

OBJECTIVE: Study the effect of loss of expression of Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, on hematopoietic stem cells (HSCs). METHODS: We examined the fetal livers of mouse embryos with homozygous disruption of the Pitx2 gene, using flow cytometry immunophenotyping analysis, as well as immunohistochemistry techniques. We further investigated the role of Pitx2 in HSCs using a chimeric mouse model system. Pitx2 null embryonic stem (ES) cell clones were generated from embryonic day 3.5 blastocysts of Pitx2 null embryos. The Pitx2 null donor ES cell contribution to the adult hematopoietic system was confirmed by identifying donor-specific glucose-phosphate isomerase isotype in the erythrocytes using cellulose acetate eletrophoresis, and by demonstrating donor-specific major histocompatibility complex antigen allotype on the granulocytes/monocytes and T and B lymphocytes of the chimeric mice using flow cytometry analysis. RESULTS: Pitx2 homozygous null fetal livers are decreased in size and overall cellularity. The erythroid cell component of these livers is further reduced as compared to that of their wild-type and heterozygous littermates. Detailed quantitative analysis of the chimeric mice revealed contribution of Pitx2 null ES cells to erythroid, myeloid, lymphoid, and megakaryocytic lineages. The quantitative level of ES cell contribution to the peripheral hematopoietic cells was proportional to the level of general chimerism as determined by coat color. CONCLUSION: Although the fetal livers of Pitx2 null embryos displayed signs of impaired erythropoiesis, Pitx2 gene disrupted HSCs can contribute to hematopoiesis under physiological conditions.     

绿色荧光蛋白标记的小鼠胚胎干细胞系的建立及向心肌细胞分化

目的建立能够稳定表达绿色荧光蛋白（green fluorescent prote in,GFP）的小鼠胚胎干细胞系,并诱导其向心肌细胞分化。方法质粒pEGFP-N1脂质体复合体转染小鼠胚胎干细胞,经G418筛选后选取GFP强阳性克隆进行扩增建系。对稳定表达GFP的胚胎干细胞系进行畸胎瘤形成检测,观察其多向分化潜能。诱导GFP阳性胚胎干细胞向心肌细胞分化。免疫荧光及RT-PCR检测心肌细胞特异性标志物。结果转染后胚胎干细胞经20次传代后仍然表达GFP,裸鼠皮下接种胚胎干细胞后3050 d均可形成畸胎瘤。GFP阳性胚胎干细胞成功向心肌细胞分化。结论成功建立表达GFP的小鼠胚胎干细胞系,并可诱导其向心肌细胞分化。     

Localization of spontaneously hyperactive B cells of NZB mice to a specific B cell subset.

NZB mice produce numerous autoantibodies and have a subpopulation of B cells characterized by marked spontaneous hypersecretion of IgM. The latter trait is determined by autosomal genes, in F1 hybrids of NZB and normal strains. We tested the hypothesis that the hypersecreting B cells of NZB mice are contained within a specific subpopulation by examining (CBA/N X NZB)F1 hybrids. CBA/N mice have an X-linked recessive defect that results in the absence of a functionally distinct B cell subpopulation and impaired antibody responses. The hyperactivity of B cells, characteristic of the NZB parent, was transmitted to the F1 female, but was not expressed by the F1 male, which manifested the CBA/N B cell hyporesponsiveness. By contrast, the NZB xenotropic virus was expressed equally by both male and female F1 mice. We conclude that the NZB B cell abnormality resides within the B cell subpopulation affected by the CBA/N mutation.     

An orally bioavailable spleen tyrosine kinase inhibitor delays disease progression and prolongs survival in murine lupus

OBJECTIVE: To assess whether R788, an orally bioavailable small molecule inhibitor of spleen tyrosine kinase (Syk)-dependent signaling, could modulate disease in lupus-prone (NZB x NZW)F1 (NZB/NZW) mice via inhibition of Fc receptor (FcR) and B cell receptor signaling. METHODS: R788 was administered to NZB/NZW mice before and after disease onset. Proteinuria, blood urea nitrogen levels, and autoantibody titers were examined periodically, and overall survival and renal pathologic features were assessed following long-term treatment (24-34 weeks). The distribution and immunophenotype of various splenic T cell and B cell subpopulations were evaluated at the time of study termination. Arthus responses in NZB/NZW mice pretreated with R788 or Fc-blocking antibody (anti-CD16/32) were also examined. RESULTS: When R788 was administered prior to or after disease onset, it delayed the onset of proteinuria and azotemia, reduced renal pathology and kidney infiltrates, and significantly prolonged survival of lupus-prone NZB/NZW mice; autoantibody titers were minimally affected throughout the study. Dose-dependent reductions in the numbers of CD4+ activated T cells expressing high levels of CD44 or CD69 were apparent in spleens from R788-treated mice. Minimal effects on the numbers of naive T cells expressing CD62 ligand and total CD8+ T cells per spleen were observed following long-term drug treatment. R788 pretreatment resulted in reduced Arthus responses in NZB/NZW mice, similar to results obtained in mice pretreated with FcR-blocking antibody. CONCLUSION: We demonstrate that a novel Syk-selective inhibitor prevents the development of renal disease and treats established murine lupus nephritis. These data suggest that Syk inhibitors may be of therapeutic benefit in human lupus and related disorders.     

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F(1) genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F(1) ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F(1) ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F(1) ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F(1) ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F(1) ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.     

Distinct effects of the recurrent Mlh1G67R mutation on MMR functions, cancer, and meiosis

Mutations in the human DNA mismatch repair (MMR) gene MLH1 are associated with hereditary nonpolyposis colorectal cancer (Lynch syndrome, HNPCC) and a significant proportion of sporadic colorectal cancer. The inactivation of MLH1 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of DNA damaging agents. To study the effect of MLH1 missense mutations on cancer susceptibility, we generated a mouse line carrying the recurrent Mlh1(G67R) mutation that is located in one of the ATP-binding domains of Mlh1. Although the Mlh1(G67R) mutation resulted in DNA repair deficiency in homozygous mutant mice, it did not affect the MMR-mediated cellular response to DNA damage, including the apoptotic response of epithelial cells in the intestinal mucosa to cisplatin, which was defective in Mlh1(-/-) mice but remained normal in Mlh1(G67R/G67R) mice. Similar to Mlh1(-/-) mice, Mlh1(G67R/G67R) mutant mice displayed a strong cancer predisposition phenotype. However, in contrast to Mlh1(-/-) mice, Mlh1(G67R/G67R) mutant mice developed significantly fewer intestinal tumors, indicating that Mlh1 missense mutations can affect MMR tumor suppressor functions in a tissue-specific manner. In addition, Mlh1(G67R/G67R) mice were sterile because of the inability of the mutant Mlh1(G67R) protein to interact with meiotic chromosomes at pachynema, demonstrating that the ATPase activity of Mlh1 is essential for fertility in mammals.     

Long-term reconstitution of the mouse hematopoietic system by embryonic stem cell-derived fetal liver.

Murine embryonic stem (ES) cells are permanent blastocyst-derived cell lines capable of contributing to a wide variety of tissues, including the germ line, after injection into host blastocysts. Recently, we have shown that ES cells can produce all of the cells of the developing fetus after aggregation with developmentally compromised tetraploid embryos. Completely ES cell-derived embryos die perinatally, but the liver of these embryos is a source of entirely ES cell-derived hematopoietic progenitors. We have taken 14- to 15-day fetal liver cells from ES cell-tetraploid chimeras and reconstituted the hematopoietic system of lethally irradiated adult recipient mice. ES cell-derived hematopoietic stem cells were capable of long-term (greater than 6 months) repopulation of irradiated recipients, and all hematopoietic cell lineages analyzed (erythrocytes, T cells, mast cells, and macrophages) were derived exclusively from ES cells in such recipients. Thus, ES cells retain the capacity to differentiate into all hematopoietic cell types after prolonged passage in culture. This approach should provide a direct route to the production of mice whose hematopoietic cells carry genetic alterations that would be lethal if passed through the germ line.     

A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development

Notch1 is known to play a critical role in regulating fates in numerous cell types, including those of the hematopoietic lineage. Multiple defects exhibited by Notch1-deficient embryos confound the determination of Notch1 function in early hematopoietic development in vivo. To overcome this limitation, we examined the developmental potential of Notch1(-/-) embryonic stem (ES) cells by in vitro differentiation and by in vivo chimera analysis. Notch1 was found to affect primitive erythropoiesis differentially during ES cell differentiation and in vivo, and this result reflected an important difference in the regulation of Notch1 expression during ES cell differentiation relative to the developing mouse embryo. Notch1 was dispensable for the onset of definitive hematopoiesis both in vitro and in vivo in that Notch1(-/-) definitive progenitors could be detected in differentiating ES cells as well as in the yolk sac and early fetal liver of chimeric mice. Despite the fact that Notch1(-/-) cells can give rise to multiple types of definitive progenitors in early development, Notch1(-/-) cells failed to contribute to long-term definitive hematopoiesis past the early fetal liver stage in the context of a wild-type environment in chimeric mice. Thus, Notch1 is required, in a cell-autonomous manner, for the establishment of long-term, definitive hematopoietic stem cells (HSCs).     

Production of medakafish chimeras from a stable embryonic stem cell line

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.     

Autoantibodies inhibit interleukin-7-mediated proliferation and are associated with the age-dependent loss of pre-B cells in autoimmune New Zealand Black Mice

Surface IgM+B220+ B cell precursors can be categorized as either leukosialin (CD43/S7) negative (late stage pre-B cells) or positive (pro-B/early pre-B cells). In autoimmune New Zealand Black (NZB) mice, bone marrow small pre-B cells (IgM-CD43-B220+) and pro-B/early pre-B cells (IgM-CD43+B220+) declined significantly with age. In particular, subpopulations of pro-B/early pre-B cells expressing the heat stable antigen (HSA) were found in lower proportions with age. Significant decreases in interleukin-7 (IL-7) colony forming units (CFU) were also seen in NZB mice by 6 to 8 months of age and accompanied alterations in the numbers of pro-B and pre-B cells in bone marrow. Concomitant with reduced numbers of B lineage precursor cells and IL-7 CFU in vivo, NZB mice produced serum IgM antibodies that strongly inhibited IL-7 CFU responses in vitro. Two monoclonal IgM antibodies (5G9, 2F5) derived from LPS stimulated 10-month-old NZB splenocytes recognized pre-B cell surface antigens on both pre-B cell lines and on IL-7 stimulated bone marrow pro-B/pre-B cells. However, these monoclonal antibodies (MoAb) failed to significantly stain ex vivo bone marrow cells. The 5G9 and 2F5 MoAbs also partially inhibited IL-7 CFU in vitro. These results suggest that NZB bone marrow becomes increasingly deficient in B cell precursors and especially in IL-7 responsive pre-B cells with age. IgM serum antibodies and monoclonal IgM antibodies derived from older NZB mice inhibit pre-B cell growth to IL-7. The production of such autoantibodies may interfere with B cell development in aging NZB mice by preventing IL-7-mediated proliferation.     

Abnormal microRNA-16 locus with synteny to human 13q14 linked to CLL in NZB mice

New Zealand black (NZB) mice with autoimmune and B lymphoproliferative disease (B-LPD) are a model for human chronic lymphocytic leukemia (CLL). A genomewide linkage scan of the NZB loci associated with lymphoma was conducted in F1 backcrosses of NZB and a control strain, DBA/2. Of 202 mice phenotyped for the presence or absence of LPD, surface maker expression, DNA content, and microsatellite polymorphisms, 74 had disease. The CD5(+), IgM(+), B220(dim), hyperdiploid LPD was linked to 3 loci on chromosomes 14, 18, and 19 that are distinct from previously identified autoimmunity-associated loci. The region of synteny with mouse D14mit160 is the human 13q14 region, associated with human CLL, containing microRNAs mir-15a16-1. DNA sequencing of multiple NZB tissues identified a point mutation in the 3' flanking sequence of the identical microRNA, mir-16-1, and this mutation was not present in other strains, including the nearest neighbor, NZW. Levels of miR-16 were decreased in NZB lymphoid tissue. Exogenous miR-16 delivered to an NZB malignant B-1 cell line resulted in cell-cycle alterations and increased apoptosis. Linkage of the mir-15a/16-1 complex and the development of B-LPD in this spontaneous mouse model suggest that the altered expression of the mir-15a/16-1 is the molecular lesion in CLL.     

Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.

We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.