Lynn Doglio

A strain-specific modifier on mouse chromosome 4 controls the methylation of independent transgene loci

A transgene, pHRD, is highly methylated in 12 independent mouse lines when in a C57BL/6 strain background, but becomes progressively less methylated when bred into a DBA/2 background. Transgenes inherited from the mother are generally more methylated; however, this parental effect disappears following continued breeding into the nonmethylating strain. Mapping experiments using BXD recombinant inbred mice as well as other inbred strains indicate that a single strain-specific modifier (Ssm-1) linked to, but distinct from, Fv-1 is responsible for the strain effect. In addition to the methylated and unmethylated transgenic phenotypes, certain mice exhibit a partial methylation pattern that is a consequence of an unusual cellular mosaicism. The pHRD transgene, containing target sequences for the V(D)J recombinase, undergoes site-specific recombination only in lymphoid tissues. This V-J joining is restricted primarily to unmethylated transgene copies.

Transgenic mice demonstrate a testis-specific promoter for lactate dehydrogenase, LDHC

The mammalian genome encodes a family of lactate dehydrogenase (LDH) isozymes. Two of these, ldha andldhb, are expressed ubiquitously. The ldhc gene is active only in the germinal epithelium during spermatogenesis. In our analysis of ldhc gene regulation, we found that a 60-base pair promoter sequence was sufficient for testis-specific expression in an in vitro transcription assay. To confirm these findings, a genomic fragment containing 100 base pairs overlapping the transcription start site was isolated and linked to theEscherichia coli lacZ gene. We report that this genomic fragment drives testis-specific expression in transgenic mice. We conclude that transcription of the transgene and possibly of the endogenous ldhc gene is restricted to leptotene/pachytene primary spermatocytes.

The endocytic recycling regulator EHD1 is essential for spermatogenesis and male fertility in mice.

BackgroundThe C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1-/- mice using a Cre/loxP system.ResultsBoth male and female Ehd1-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1-/- adult males were infertile and displayed decreased testis size, whereas Ehd1-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1-/- mice.ConclusionsContrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.

U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia

Upregulated gene 19 (U19)/ELL-associated factor 2 (Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 deficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

GLI1 Localization in the Germinal Epithelial Cells Alternates Between Cytoplasm and Nucleus: Upregulation in Transgenic Mice Blocks Spermatogenesis in Pachytene

Abstract The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice.

The four highly homologous members of the C‐terminal EH domain‐containing (EHD) protein family (EHD1‐4) regulate endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4−/− mice confirmed EHD4 deletion. Ehd4−/− mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (Day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (Day 31) were seen in the Ehd4−/− testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium, and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4−/− males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and EHD4 levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age‐dependent manner. We conclude that high levels of EHD1 in the adult Ehd4−/− testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of postmitotic and postmeiotic germ cells and implicate EHD protein‐mediated endocytic recycling as an important process in germ cell development and testis function. genesis 48:328–342, 2010.

Role of Mammalian Ecdysoneless in Cell Cycle Regulation

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecdlox/lox mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecdlox/lox mouse embryonic fibroblasts led to a proliferative block due to a delay in G1-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb at the pocket domain and competed with E2F for binding to hypophosphorylated Rb. Our results demonstrate that mammalian Ecd plays a role in cell cycle progression via the Rb-E2F pathway.

Expression of λ and K Genes Can Occur in all B Cellsand is Initiated Around the Same Pre-B-CellDevelopmental Stage

Transgenic mice that carry a λ2 transgene under the control of the Vλ2 promoter and the Eλ2-4 enhancer (λ2Eλ mice) are described. A high proportion of B cells in the spleen and the bone marrow express the λ transgene on the cell membrane. λ2 protein is synthesized by all λ2Eλ-derived spleen B-cell hybridomas that have retained the transgene, suggesting that all B cells have the ability to express λ genes. Feedback inhibition of endogenous K-gene rearrangement is significant, but not complete. The results are similar to those with transgenic mice expressing the same λ2 transgene under the control of the heavy-chain enhancer (λ2EH mice). Although the λ2EH transgene is expressed before the λ2Eλ transgene, feedback inhibition seems to occur at about the same stage of B-cell development, regardless of the timing of expression of the λ transgenes. Apparently, feedback is not necessarily coincident with the assembly of a heavy-chain/light-chain complex in pre-B cells. Expression of λ in the normal fetal liver coincides with the expression of K; thus, it appears that λ-gene transcription is not delayed. The differential rearrangement of K and λ genes is discussed in the light of these findings.

Enucleation of feeder cells and egg cells with psoralens

The cell nucleus must be inactivated or destroyed in order to generate feeder layers for cultured cells or to prepare recipient egg cells for nuclear transfer. Existing enucleation techniques are either cumbersome or employ toxic chemicals. Here we report a new method to enucleate cells by treatment with a psoralen and long‐wave ultraviolet light. The technique is >90% efficient and causes little cytoplasmic damage to the treated cell. We have used psoralen treatment to enucleate a wide variety of cells, including eggs, sperm, HeLa cells, and fibroblasts. Colonies of human embryonic stem cells (hESCs) and human keratinocyte precursors grown on psoralen‐treated feeders are indistinguishable from those grown on gamma‐irradiated or mitomycin C‐treated cells. Psoralen enucleation provides a rapid, simple, and non‐toxic method to generate feeder cells. The technique is also useful for nuclear transfer studies in species with large eggs whose cleavage divisions are not regulated by cell‐cycle checkpoints. Developmental Dynamics 238:2614–2621, 2009.

Rearrangement and Expression of Immunoglobulin Genes in Transgenic Mice

Transgenic mice are discussed which carry a rearrangement test transgene. The methylation status of the transgene varies, depending on the background mouse strain. When the transgene is bred into the C57BL/6 strain, it is completely methylated and not rearranged in lymphoid organs. After several generations of crossing into DBA/2 or SJL the transgene becomes unmethylated and rearranges at high frequency. A strain specific modifier of DNA methylation (Ssm-1) was mapped close to the Friend virus susceptibility locus (Fv-1) on mouse chromosome 4. Rearranged transgenes from spleen, bone marrow and thymus of adult mice or fetal liver were cloned and sequenced. A great variety of joints was found, with about 1/3 being in the correct reading frame. Small deletions into the V- and J-coding ends as well as N region additions contributed to the variability. The fetal joints showed no N regions. Since no functional immunoglobulin (Ig) gene can be created from this artificial test gene, the data indicate that the rearrangement mechanism of the fetus differs from that of the adult.