Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.

Microinjection of foreign DNA into fertilized mammalian eggs is a convenient means of introducing genes into the germ line. Some of the more important parameters that influence successful integration of foreign DNA into mouse chromosomes are described. The effects of DNA concentration, size, and form (supercoiled vs. linear with a variety of different ends) are considered as well as the site of injection (male pronucleus, female pronucleus, or cytoplasm) and buffer composition. The optimal conditions for integration entail injection of a few hundred linear molecules into the male pronucleus of fertilized one-cell eggs. Under these conditions about 25% of the mice that develop inherit one or more copies of the microinjected DNA. The overall efficiency also depends on the choice of mouse strains; for example, generating transgenic mice that express foreign growth hormone genes is about eight times easier with C57/BL6 X SJL hybrid mice than with inbred C57/BL6 mice.     

Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17;18 translocation.

Integrated hepatitis B virus (HBV) DNA is almost invariably found in hepatocellular carcinomas (HCC) which develop in HBV carriers. Integrated HBV DNAs from two single-integration HCCs (C3 and C4) have been cloned, and the cellular integration sites have been analyzed. Integrated HBV DNA of C3 is present in chromosome 6 and contains a nearly complete linear HBV genome. The HBV DNA integration in tumor C3 was not associated with major rearrangements of cellular DNA. In contrast, the integrated HBV DNA in C4 contains a large inverted repeat of HBV DNA, in which each repeat consists of a linear HBV DNA segment similar to that present in C3. The C4 integration was also accompanied by a cellular DNA translocation at the HBV integration site. The translocation occurred between chromosomes 17 and 18, along with a deletion of at least 1.3 kilobases of chromosome 18 DNA at the translocation site. Our data support a model in which postintegration rearrangement of integrated HBV and cellular DNA results in the generation of chromosomal aberrations. These chromosomal aberrations may function in a multistage mechanism leading to fully malignant HCC.     

High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize

Somatic chromosome spreads from maize (Zea mays L.) plants containing B-A translocation chromosomes undergoing the chromosome type breakage-fusion-bridge cycle were examined by FISH. The size and type of extra chromosomes varied among cells of the same individual. A collection of minichromosomes derived from the chromosome type breakage-fusion-bridge cycle was examined for the presence of stable dicentric chromosomes. Six of 23 chromosomes in the collection contained two regions with DNA sequences typical of centromeres. Functional analysis and immunolabeling of CENH3, the centromere-specific histone H3 variant, revealed only one functional centromere per chromosome, despite the duplicate centromere sequences. One plant was found with an inactive B centromere that had been translocated to the short arm of chromosome 9. The translocated centromere region appeared identical to that of a normal B chromosome. The inactivation of the centromeres was stable for at least four generations. By using dicentrics from dispensable chromosomes, centromere inactivation was found to be quite common under these circumstances.     

Construction and characterization of genomic libraries from specific human chromosomes.

Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.     

Genetic analysis of the 15;17 chromosome translocation associated with acute promyelocytic leukemia.

Somatic cell hybrids have been constructed between a thymidine kinase-deficient mouse cell line and blood leukocytes from a patient with acute promyelocytic leukemia showing the 15q+;17q- chromosome translocation frequently associated with this disease. One hybrid contains the 15q+ translocation chromosome and very little other human material. We have shown that the c-fes oncogene, which has been mapped to chromosome 15, is not present in this hybrid and, therefore, probably is translocated to the 17q- chromosome. Analysis of the genetic markers present in this hybrid has enabled a more precise localization of the translocation breakpoints on chromosomes 15 and 17. Our experiments also have enabled an ordering and more precise mapping of several genetic markers on chromosomes 15 and 17.     

Insertion of a bacterial gene into the mouse germ line using an infectious retrovirus vector.

Using a Moloney leukemia virus vector containing the bacterial neo gene, we demonstrate that retrovirus vectors can be used to introduce genes into the mouse germ line. Infection of preimplantation embryos with the vector MLV-NEO.1 resulted in integration of neo sequences in approximately equal to 10% of the progeny mice. One of these animals, mouse F.2, contained approximately six MLV-NEO.1 proviruses at independent integration sites, each present at less than a single copy per cell. This mosaic mouse transmitted one of these proviruses to her offspring, producing a line of transgenic mice carrying a full-length, unrearranged MLV.NEO.1 provirus at a single chromosomal integration site. Mice homozygous at this MLV-NEO.1 locus have also been produced. No expression of the neo gene has been detected in the transgenic mice, either by screening of primary bone marrow or lung cells for resistance to G418 or by RNA transfer blot analysis of RNA from several tissues. In addition, the neo gene was found to be extensively methylated in the transgenic mice; however, treatment of primary cells with 5-azacytidine did not induce G418 resistance. The inactivity of the MLV-NEO.1 provirus in transgenic mice and potential means of eliciting neo expression under these conditions are discussed.     

v-abl activates embryonic globin gene expression in mouse erythroleukemia cells.

The Philadelphia chromosome translocation, which is present in 90-95% of chronic myelogenous leukemia patients, involves translocation of the c-abl protooncogene to chromosome 22 and is accompanied by activation of embryonic globin gene expression in the K562 chronic myelogenous leukemia cell line. To test directly if the protein products of the translocated c-abl protooncogene can activate embryonic globin gene expression, we transfected the v-abl oncogene (which shares the property of autophosphorylation with the translocated c-abl protooncogene) into mouse erythroleukemia cells. v-abl-transfected mouse erythroleukemia cells, which contained multiple copies of the v-abl transgenome, exhibited activation of mouse embryonic globin gene expression. These results suggest that the translocated c-abl protooncogene of the Philadelphia chromosome translocation is central to the pathogenesis of chronic myelogenous leukemia and that it may result in the activation of embryonic globin genes in some chronic myelogenous leukemia cell lines.     

FVB/N: an inbred mouse strain preferable for transgenic analyses.

FVB/N mice offer a system suitable for most transgenic experiments and subsequent genetic analyses. The inbred FVB/N strain is characterized by vigorous reproductive performance and consistently large litters. Moreover, fertilized FVB/N eggs contain large and prominent pronuclei, which facilitate microinjection of DNA. The phenotype of large pronuclei in the zygote is a dominant trait associated with the FVB/N oocyte but not the FVB/N sperm. In experiments to generate transgenic mice, the same DNA constructs were injected into three different types of zygotes: FVB/N, C57BL/6J, and (C57BL/6J x SJL/J)F1. FVB/N zygotes survived well after injection, and transgenic animals were obtained with efficiencies similar to the F1 zygotes and much better than the C57BL/6J zygotes. Genetic markers of the FVB/N strain have been analyzed for 44 loci that cover 15 chromosomes and were compared with those of commonly used inbred strains. In addition to the albino FVB/N strain, pigmented congenic strains of FVB/N are being constructed. These features make the FVB/N strain advantageous to use for research with transgenic mice.     

Transgenic rescue of GATA-1-deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia

Association of GATA-1 and its cofactor Friend of GATA-1 (FOG-1) is essential for erythroid and megakaryocyte development. To assess functions of GATA-1-FOG-1 association during mouse development, we used the GATA-1 hematopoietic regulatory domain to generate transgenic mouse lines expressing a mutant GATA-1, which contains a substitution of glycine 205 for valine (V205G) that abrogates its association with FOG-1. We examined whether the transgenic expression of mutant GATA-1 rescues GATA-1 germ line mutants from embryonic lethality. In high-expressor lines we observed that the GATA-1(V205G) rescues GATA-1-deficient mice from embryonic lethality at the expected frequency, revealing that excess GATA-1(V205G) can eliminate the lethal anemia that is due to GATA-1 deficiency. In contrast, transgene expression comparable to the endogenous GATA-1 level resulted in much lower frequency of rescue, indicating that the GATA-1-FOG-1 association is critical for normal embryonic hematopoiesis. Rescued mice in these analyses exhibit thrombocytopenia and display dysregulated proliferation and impaired cytoplasmic maturation of megakaryocytes. Although anemia is not observed under steady-state conditions, stress erythropoiesis is attenuated in the rescued mice. Our findings reveal an indispensable role for the association of GATA-1 and FOG-1 during late-stage megakaryopoiesis and provide a unique model for X-linked thrombocytopenia with inherited GATA-1 mutation.     

Specific chromosomal abnormalities characterize fibrosarcomas of bovine papillomavirus type 1 transgenic mice.

In the BPV1.69 line of transgenic mice, the bovine papillomavirus type 1 genome elicits both benign dermal fibroblastic proliferation (fibromatoses) and malignant fibrosarcomas. Because these lesions arise only with time, nonviral factors appear to be involved. We have karyotyped several primary tumors as well as a series of low-passage cell lines derived both from fibromatoses and from fibrosarcomas. The fibrosarcomas, but not the preneoplastic fibromatoses, show consistent abnormalities of one or both of two chromosomes, chromosome 8 (trisomy or duplication) and chromosome 14 (monosomy or translocation). The chromosomal abnormalities are not a direct consequence of the viral integration, which we have mapped to chromosome 15 by in situ hybridization. These results suggest that transgenic mice can be used to study the role(s) of cytogenetic changes in tumorigenesis and may direct the search for genes involved in tumor progression.     

Analysis of the human alpha-globin gene cluster in transgenic mice.

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-globin genes is not equivalent to that upstream of the beta locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.     

Clonal development and karyotype evolution during leukemogenesis of BCR/ABL transgenic mice.

The Philadelphia (Ph) translocation is responsible for the generation of the chimeric BCR/ABL oncogene. The Ph chromosome constitutes the earliest detectable chromosome abnormality in chronic myelogenous leukemia and is also found in acute lymphoblastic leukemia. Mice transgenic for a P190 BCR/ABL-producing DNA construct develop lymphoblastic leukemia/lymphoma and provide an opportunity to study early stages of the disease as well as progression. In this study, we have karyotyped the bone marrow of 10 19-day-old BCR/ABL P190 transgenic mice from a line that reproducibly develops leukemia/lymphoma. Leukemic cells from 17 terminally ill transgenic founders and progeny were also karyotyped as well as bone marrow transplant recipients of leukemic donor marrow. Karyotypically visible aberrations were absent from the early stages of BCR/ABL P190-generated leukemia and normal metaphases could be found even in the terminal stages of the disease. A high frequency of aneuploidy was found in advanced leukemia, with a marked preference for the gain of mouse chromosomes 12, 14, or 17. These results point to a primary role for BCR/ABL in leukemogenesis and suggest a destabilizing effect of the BCR/ABL gene on the regulation of cell division.     

Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice

The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases, generates the AML1/ETO fusion protein. To study the role of AML1/ETO in the pathogenesis of AML, we used the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1/ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1/ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1(+/AML1-ETO ires EGFP) (abbreviated Sca(+/AE)) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca(+/AE) mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca(+/AE) mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1/ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.     

Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice.

Two lines of transgenic mice with one to two copies of a DNA fragment containing nucleotides -364 to +45 of the murine alpha A-crystallin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene expressed the CAT gene only in their eye lenses. Both CAT activity and alpha A-crystallin were first detected in eyes at approximately 12.5 days of embryonic development, suggesting that the alpha A-CAT fusion gene and the endogenous alpha A-crystallin gene are co-regulated during lens development in the transgenic mice. These experiments show that the murine alpha A-crystallin gene contains a short, cis-acting, tissue-specific regulatory sequence at its 5' end that can target the expression of the bacterial CAT gene, and probably foreign eukaryotic genes, specifically to the ocular lens.     

Targeted integration of foreign DNA into a defined locus on chromosome 19 in K562 cells using AAV-derived components

Targeted integration of foreign DNA is ideal for gene therapy, particularly when target cells such as hematopoietic cells actively divide and proliferate. Adeno-associated virus (AAV) has been shown to integrate its genome into a defined locus, AAVS1 (19q13.3-qter). The inverted terminal repeat (ITR) and Rep proteins are responsible for this site-specific integration, and a system has been developed that delivers a gene preferentially into AAVS1 by using these components of AAV. We examined whether this system could be applied to gene transfer into K562 cells. Two rep expression plasmids were tested, 1 driven by the cytomegalovirus (CMV) promoter (pCMVR78) and the other under the translational control of an internal ribosome entry site (pMGiR78) with mouse mammary tumor virus promoter. K562 cells were cotransfected with a rep plasmid and a plasmid containing a neo gene flanked by the ITRs. G418-resistant clones were isolated and analyzed by Southern blot analysis and fluorescence in situ hybridization (FISH). Southern blot analysis suggested AAVS1-specific integration of the neo gene in 6 (35%) of 17 clones when K562 cells were transfected with pMGiR78 by lipofection. FISH located the neo gene on chromosome 19 in 5 of these 6 clones (29%). Eight (32%) of 25 clones obtained by electroporation with pCMVR78 had the neo gene at AAVS1, according to Southern blot analysis, and 4 of these 8 clones (16%) were positive according to FISH analysis. These results suggest that site-specific integration of foreign DNA can be achieved at a significantly high rate in human hematopoietic cells using the AAV components.     

Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2

We have previously shown that the putative mammalian retromer components sorting nexins 1 and 2 (Snx1 and Snx2) result in embryonic lethality when simultaneously targeted for deletion in mice, whereas others have shown that Hbeta58 (also known as mVps26), another retromer component, results in similar lethality when targeted for deletion. In the current study, we address the genetic interaction of these mammalian retromer components in mice. Our findings reveal a functional interaction between Hbeta58, SNX1, and SNX2 and strongly suggest that SNX2 plays a more critical role than SNX1 in retromer activity during embryonic development. This genetic evidence supports the existence of mammalian retromer complexes containing SNX1 and SNX2 and identifies SNX2 as an important mediator of retromer biology. Moreover, we find that mammalian retromer complexes containing SNX1 and SNX2 have an essential role in embryonic development that is independent of cation-independent mannose 6-phosphate receptor trafficking.     

Identification of cooperative genes for NUP98-HOXA9 in myeloid leukemogenesis using a mouse model

The chromosomal translocation t(7; 11)(p15;p15), observed in human myeloid leukemia, results in a NUP98 and HOXA9 gene fusion. We generated a transgenic mouse line that specifically expressed the chimeric NUP98-HOXA9 gene in the myeloid lineage. While only 20% of the transgenic mice progressed to leukemia after a latency period, myeloid progenitor cells from nonleukemic transgenic mice still exhibited increased proliferative potential. This suggested that the NUP98-HOXA9 fusion induced a preleukemic phase, and other factors were required for complete leukemogenesis. NUP98-HOXA9 expression promoted the onset of retrovirus-induced BXH2 myeloid leukemia. This phenomenon was used to identify cooperative disease genes as common integration sites (CISs). Meis1, a known HOX cofactor, was identified as a CIS with a higher integration frequency in transgenic than in wild-type BXH2 mice. By the same means we identified further 4 candidate cooperative genes, Dnalc4, Fcgr2b, Fcrl, and Con1. These genes cooperated with NUP98-HOXA9 in transforming NIH 3T3 cells. The system described here is a powerful tool to identify cooperative oncogenes and will assist in the clarification of the multistep process of carcinogenesis.