No abstract

A method of gene targeting that allows the inducible inactivation of a target gene in mice is presented. The method uses an interferon-responsive promoter to control the expression of Cre recombinase. Here, Cre was used to delete a segment of the DNA polymerase beta gene flanked by IoxP recombinase recognition sites. Deletion was complete in liver and nearly complete in lymphocytes within a few days, whereas partial deletion was obtained in other tissues. This method can be used for the inducible inactivation of any other gene in vivo.

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.

We have characterized the progressive stages of chronic intestinal inflammation that develops spontaneously in specific pathogen-free (SPF) mice with a targeted disruption in the IL-10 gene (IL-10 Ϫ / Ϫ ). Our longitudinal studies showed that inflammatory changes first appear in the cecum, ascending and transverse colon of 3-wk-old mutants. As the disease progressed, lesions appeared in the remainder of the colon and in the rectum. Some aged IL-10 Ϫ / Ϫ mice also developed inflammation in the small intestine. Prolonged disease with transmural lesions and a high incidence of colorectal adenocarcinomas (60%) was observed in 6-mo-old mutants. Mechanistic studies have associated uncontrolled cytokine production by activated macrophages and CD4

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as, a direct miR-7 target, was enhanced in -deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the non-homologous end joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA Ligase IV by gene silencing, the Ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a `traffic light´ and other reporter systems. Suppression of KU70 and DNA Ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to 8-fold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.The clustered, regularly interspaced, short palindromic repeats (CRISPRs)-Cas9 endonuclease represent a versatile tool for genome engineering, enabling the induction of site-specific genomic double-strand breaks (DSBs) by single guide RNAs (sgRNAs) 1 .In mammalian cells DSBs are mostly repaired by the non-homologous end joining (NHEJ) pathway 2,3 , frequently leading to the loss of nucleotides from the DSB ends. This enables the efficient construction of knockout alleles through the induction of frameshift To quantitatively determine the outcome of CRISPR-induced DSB repair, we first generated human HEK293 cells with a `traffic light´ reporter 7 (TLR) vector integrated into the AAVS1 locus 8 (Fig. 1a). HEK293 cells were transfected with an AAVS1 targeting vector carrying the TLR insert and Cas9 and AAVS1-specific sgRNA expression plasmids ( Supplementary Fig. 1 , and up to 7-fold by the Ad4 protein pair ( Fig. 2c and Supplementary Fig. 2.2).Titration of SCR7 on AAVS1 TLR/+ cells showed an optimal effect at 1 µM concentration ( Supplementary Fig. 2.3). In the presence of two shRNAs, SCR7 or Ad4 proteins we noticed diminished fluorescence signals within the population of Venus + cells at 72h after transfection (Fig. 2a, Supplementary Fig. 2.1, 2.3c), indicating reduced Venus expression in cells undergoing NHEJ blockade, possibly caused by local chromatin remodeling through an extended DNA damage response 13,14 . However, Venus expression was normal in clones established from AAVS1 TLR/+ cells targeted in the presence of Ad4 proteins, indicating that this effect is only transient (Supplementary Fig. 2.3d). From the sample expressing the Ad4 proteins, Venus + cells were sorted, and we established 24 clones to confirm the integrity of the repaired TLR loci using PCR and sequence analysis (Supplementary Table 2). In contrast to the increase of Venus + cells, RFP + cells decreased from 3% in the controls to 1.7%, 1.4% or 0.6% in the presence of 5 shRNAs, SCR7 or Ad4 proteins, respectively (Fig. 2a, 2b). Whether the residual NHEJ activity relies on the KU/Ligase IV independent alternative end-joining mechanism 15 rem...

We propose a method for combining QCD matrix elements and parton showers in Monte Carlo simulations of hadronic final states in e + e − annihilation. The matrix element and parton shower domains are separated at some value y ini of the jet resolution, defined according to the k T -clustering algorithm. The matrix elements are modified by Sudakov form factors and the parton showers are subjected to a veto procedure to cancel dependence on y ini to next-to-leading logarithmic accuracy. The method provides a leading-order description of hard multi-jet configurations together with jet fragmentation, while avoiding the most serious problems of double counting. We present first results of an approximate implementation using the event generator APACIC++.

Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system also depends on BCR expression, we have generated a transgenic mouse in which the BCR can be inducibly ablated through V region gene deletion. Ablation leads to rapid death of mature B lymphocytes, which is preceded by down-regulation of MHC antigens and up-regulation of CD95 (Fas) and can be delayed by constitutive bcl-2 expression.