Germ Line Deletion Reveals a Nonessential Role of Atypical Mitogen-Activated Protein Kinase 6/Extracellular Signal-Regulated Kinase 3.

Mitogen-activated protein kinase 6/extracellular signal-regulated kinase 3 (MAPK6/ERK3) is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary dysfunction and by defects in function, activation, and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon 3 flanked by loxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing the first evidence for the existence of the ERK3/MK5 signaling complex in vivo In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile and do not display pulmonary hypoplasia, acute respiratory failure, abnormal T-cell development, reduction of thymocyte numbers, or altered T-cell selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality and lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin resistance cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal, suggesting redundant functions of both protein kinases.

Aberrant regulation of imprinted gene expression in Gtl2lacZ mice.

The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.

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.

Skeletal dysplasias, growth retardation, reduced postnatal survival, and impaired fertility in mice lacking the SNF2/SWI2 family member ETL1.

The mouse Etl1 gene encodes a nuclear protein belonging to the rapidly growing SNF2/SWI2 family. Members of this family are related to helicases and nucleic-acid-dependent ATPases and have functions in essential cellular processes such as transcriptional regulation, maintenance of chromosome stability and various aspects of DNA repair. The ETL1 protein is expressed from the two-cell stage onwards, throughout embryogenesis in a dynamic pattern with particularly high levels in the thymus, epithelia and the nervous system and in most adult tissues. As a first step to address the role of ETL1 in cells and during development, we inactivated the gene by homologous recombination. ES cells and mice lacking detectable ETL1 protein were viable, indicating that ETL1 is not essential for cell survival or for embryonic development. However, mutant mice showed retarded growth, peri/post natal lethality, reduced fertility and various defects in the sternum and vertebral column. Expressivity and penetrance of all observed phenotypes were influenced by the genetic background. Isogenic 129Sv(Pas) mice lacking ETL1 had a severely reduced thoracic volume, which might lead to respiratory failure and could account for the high incidence of perinatal death on this genetic background.

The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA.

We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

Gtl2lacZ, an insertional mutation on mouse chromosome 12 with parental origin-dependent phenotype.

We have produced a transgenic mouse line, Gtl2lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed.

The Etl-1 gene encodes a nuclear protein differentially expressed during early mouse development.

Recently, we isolated a novel mouse gene, Etl-1 (Enhancer-trap-locus-1), whose deduced amino acid sequence shows in its C-terminal portion striking homology to the brahma protein (BRM), a transcriptional regulator of homeotic genes in Drosophila, and to SNF2/SWI2, a transcriptional regulator of various genes in Saccharomyces cerevisiae. Here we report the generation of antibodies against the Etl-1 gene product (ETL-1) and describe the subcellular localization as well as the expression and distribution of the ETL-1 protein during mouse pre- and early post-implantation development. ETL-1 is a nuclear protein and is expressed in a biphasic manner during early embryogenesis. Moderate levels of ETL-1 were detected in unfertilized and fertilized eggs but in the latter the protein was not concentrated in the pronuclei and seemed evenly distributed throughout the cytoplasm. In two-cell embryos nuclear ETL-1 protein accumulated transiently and levels decreased during subsequent cleavage development. After the morula stage, ETL-1 levels increased again; in blastocysts high levels of ETL-1 were present in inner cell mass cells whereas trophectoderm cells contained little or no ETL-1. During subsequent development essentially all cell types except parietal endoderm and trophoblast cells contained high levels of ETL-1. Our results imply that nuclear ETL-1 is dispensable for the progression to the two cell stage, and suggest that during cleavage ETL-1 might be needed at the onset of embryonic transcription. In blastocysts ETL-1 function might be specifically required in cells of the inner cell mass and later in most cells of the embryo proper and extraembryonic ectoderm lineage.