Nuclear spin selective laser control of rotational and torsional dynamics

We explore the possibility of controlling rotational-torsional dynamics of non-rigid molecules with strong, non-resonant laser pulses and demonstrate that transient, laser-induced torsional alignment depends on the nuclear spin of the molecule. Consequently, nuclear spin isomers can be manipulated selectively by a sequence of time-delayed laser pulses. We show that two pulses with different polarization directions can induce either overall rotation or internal torsion, depending on the nuclear spin.Nuclear spin selective control of the angular momentum distribution may open new ways to separate and explore nuclear spin isomers of polyatomic molecules

Conditional gene trapping using the FLEx system.

The knowledge about the complete genome sequences of mouse, human, and other organisms is only the first step toward the functional annotation of all genes. It facilitates the recognition of sequence conservation, which helps to distinguish between important and not important and also coding from noncoding sequence. Nevertheless, approximately only 50% of all mouse genes have been entirely annotated to date. In the postgenomic era, large-scale projects have been initiated to describe also the expression (Emap, Eurexpress) and the function (International Gene Trap Consortium, Eucomm, Norcomm, Komp) of all mouse genes. By building up on these resources, the average amount of time starting from a gene-coding sequence to finally studying its function in a living organism or embryo, has shortened significantly within the last decade. Several recent developments, namely, in bioinformatics and gene synthesis but also in targeted and random mutagenesis have contributed to the current status. This chapter will highlight the milestones that have been undertaken in order to saturate the mouse genome with gene trap mutations. We have no intention to cover the entire field but will instead focus on most recent vectors and protocols, which have turned out to be most useful in order to promote the technology. Therefore, we apologize upfront to the many studies that could not be mentioned here solely owing to space limitations but which nevertheless made significant contributions to our current understanding. This chapter will finally provide guidance on possible uses of conditional gene trap alleles as well as detailed protocols for the application of this recent technology

Genotyping gene-trap mutant mice by real-time PCR.

A major task in the second phase of the genome sequencing projects is the identification of coding sequence within the three billion base pairs of each the mouse and human genomes. At present, in addition to computer-aided programs, several high-throughput mutagenesis programs are being undertaken worldwide in order to achieve this goal (Ref. 1). Gene-trap mutagenesis screens take advantage of random insertions into transcription units to drive a selection–reporter cassette and simultaneously to mutate the tagged genes. One of the advantages of gene-trap mutagenesis is that no a priori knowledge is needed about the structure of the tagged gene. However, because most gene-trap vectors contain splice-acceptor or splice-donor sites to capture translated gene sequence, the precise location of vector integration within introns is not known. Therefore, it is often difficult to generate external probes for Southern blots or external primers for PCR analysis in order to distinguish between homozygous and heterozygous mutants. To overcome this serious limitation in high-throughput mutagenesis screens, we developed a real-time PCR strategy that allows us to discriminate between mutants with either one or two copies of the gene-trap vector inside their genomes. Real-time quantitative PCR is based on the quantification of a fluorescent dye [5′-6-carboxyfluorescein (5′-FAM)] that is quenched by 3′-6-carboxy-tetramethylrhodamine (3′ TAMRA) when attached to a probe located between two PCR primers but is activated by the 5′ exonuclease activity of the Taq DNA polymerase (Ref. 2,3 ). Here, we describe a rapid method based on the quantification of a gene-trap vector relative to a standard locus within the mouse genome. This method allows the rapid genotyping of any gene-trap animal without prior knowledge of the mutated genes. Here, as an example, we describe the genotyping of a PT1βgeo insertion (Ref. 4) into the mouse Neurochondrin gene (Ref. 5) by comparing a multiplex PCR assay and a novel real-time-PCR-based method

Expression of neurochondrin in the developing and adult mouse brain.

Here we describe a detailed analysis of the expression of neurochondrin (ncdn) in the developing and adult mouse brain. Ncdn is first expressed in the hindbrain and spinal cord at embryonic day 10.5 (E10.5) followed by expression in the midbrain at E11.5. By E18 ncdn is also expressed in the diencephalon and telencephalon. However, strongest expression is still observed in the hindbrain. In adults, the expression in the forebrain is as strong as in the hindbrain. Ncdn is highly expressed in the hippocampus, piriform cortex, septum, amygdaloid complex, medial geniculate nucleus, inferior colliculus, cerebellar nuclei and the nuclei of the Vth, VIIth, and XIIth cranial nerves

Progressive loss of the spongiotrophoblast layer of Birc6/Bruce mutants results in embryonic lethality.

We have generated a mouse line with a mutant allele of the mouse Bruce/Birc6 gene induced by gene trap mutagenesis. Based on its structural features, Bruce is a member of the family of apoptosis inhibitor proteins (IAPs). This mutation leads to a truncated transcript and protein and results in a complete loss of the wildtype Bruce protein. Bruce mutant mice die from a progressive loss of their placental spongiotrophoblast layer between day 11.5 and 14.5 of embryonic development. The cause of the Bruce homozygous mutant phenotype is a lack of proliferation of spongiotrophoblast cells in the developing placenta. In contrast to in vitro data, which indicate a function for Bruce in apoptosis inhibition, the in vivo results presented here suggest instead a role for Bruce in cell division

HIV-1 replication in human immune cells is independent of TAR DNA binding Protein 43 (TDP-43) expression.

The TAR DNA binding protein (TDP-43) was originally identified as a host cell factor binding to the HIV-1 LTR and thereby suppressing HIV-1 transcription and gene expression (Ou et al., J.Virol. 1995, 69(6):3584). TDP-43 is a global regulator of transcription, can influence RNA metabolism in many different ways and is ubiquitously expressed. Thus, TDP-43 could be a major factor restricting HIV-1 replication at the level of LTR transcription and gene expression. These facts prompted us to revisit the role of TDP-43 for HIV-1 replication. We utilized established HIV-1 cell culture systems as well as primary cell models and performed a comprehensive analysis of TDP-43 function and investigated its putative impact on HIV-1 gene expression. In HIV-1 infected cells TDP-43 was neither degraded nor sequestered from the nucleus. Furthermore, TDP-43 overexpression as well as siRNA mediated knockdown did not affect HIV-1 gene expression and virus production in T cells and macrophages. In summary, our experiments argue against a restricting role of TDP-43 during HIV-1 replication in immune cells

The REST remodeling complex protects genomic integrity during embryonic neurogenesis.

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation

Musashi 2 is a regulator of the HSC compartment identified by a retroviral insertion screen and knockout mice.

We used a retroviral integration screen to search for novel genes that regulate HSC function. One of the genes that conferred HSC dominance when overexpressed due to an adjacent retroviral insertion was Musashi 2 (Msi2), an RNA-binding protein that can act as a translational inhibitor. A gene-trap mouse model that inactivates the gene shows that Msi2 is more highly expressed in long-term (LT) and short-term (ST) HSCs, as well as in lymphoid myeloid primed progenitors (LMPPs), but much less in intermediate progenitors and mature cells. Mice lacking Msi2 are fully viable for up to a year or more, but exhibit severe defects in primitive precursors, most significantly a reduction in the number of ST-HSCs and LMPPs and a decrease in leukocyte numbers, effects that are exacerbated with age. Cell-cycle and gene-expression analyses suggest that the main hematopoietic defect in Msi2-defective mice is the decreased proliferation capacity of ST-HSCs and LMPPs. In addition, HSCs lacking Msi2 are severely impaired in competitive repopulation experiments, being overgrown by wild-type cells even when mutant cells were provided in excess. Our data indicate that Msi2 maintains the stem cell compartment mainly by regulating the proliferation of primitive progenitors downstream of LT-HSCs