Simple and highly efficient BAC recombineering using galK selection

Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (λ) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive λ prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors

Children’s distal forearm fractures:a population-based epidemiology study of 4,316 fractures

AIMS: The aim of this study was to report a complete overview of both incidence, fracture distribution, mode of injury, and patient baseline demographics of paediatric distal forearm fractures to identify age of risk and types of activities leading to injury. METHODS: Population-based cohort study with manual review of radiographs and charts. The primary outcome measure was incidence of paediatric distal forearm fractures. The study was based on an average at-risk population of 116,950. A total number of 4,316 patients sustained a distal forearm fracture in the study period. Females accounted for 1,910 of the fractures (44%) and males accounted for 2,406 (56%). RESULTS: The overall incidence of paediatric distal forearm fractures was 738.1/100,000 persons/year (95% confidence interval (CI) 706/100,000 to 770/100,000). Female incidences peaked with an incidence of 1,578.3/100,000 persons/year at age ten years. Male incidence peaked at age 13 years, with an incidence of 1,704.3/100,000 persons/year. The most common fracture type was a greenstick fracture to the radius (48%), and the most common modes of injury were sports and falls from ≤ 1 m. A small year-to-year variation was reported during the five-year study period, but without any trends. CONCLUSION: Results show that paediatric distal forearm fractures are very common throughout childhood in both sexes, with almost 2% of males aged 13 years sustaining a forearm fracture each year. Cite this article: Bone Jt Open 2022;3(6):448–454

Domain-specific regulation of cerebellar morphogenesis by Zfp423 / ZNF423, a gene implicated in Joubert syndrome and cerebellar vermis hypoplasia

The Zfp423 gene encodes a 30-Zn-finger transcription factor that acts as a scaffold for the assembly of complex transcriptional and cellular machineries regulating neural development. While null Zfp423 mutants feature a sharp decrease in the total number of cerebellar Purkinje cells (PCs), the underlying mechanisms remain unclear. Mutations of the human homolog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), associated with other signs of classical ciliopathy outside the central nervous system. To further characterize the role of ZFP423 in cerebellar neurogenesis, we have performed morphological, cellular and molecular studies on two mutant mouse lines carrying allelic in-frame deletions of Zfp423. While both lines exhibit cerebellar hypoplasia, considerable differences are observed between the two mutants, with respect to neural progenitor differentiation, cell survival and morphogenesis. The results of this in vivo and in vitro structure-function analysis point to domain- and context-specific roles played by ZFP423 in different aspects of cerebellar development, and contribute to our understanding of its role as a disease / modifier gene in JS, CVH and other ciliopathies

Zinc-finger and helix-loop-helix transcription factors regulate Purkinje neuron neurogenesis and cerebellar corticogenesis

Many regulatory genes have been pinpointed as orchestrators of cerebellar development, from the onset of neurogenesis to the patterning of the adult cerebellar cortex, with a special reference to the development of cerebellar Purkinje cells (PCs). PCs provide the sole output from cerebellar cortical circuits, where each PC integrates myriads of presynaptic inputs, both inhibitory and excitatory. In the murine cerebellar primordium PCs are generated from a pool of ventricular zone progenitors facing the fourth ventricle between embryonic day (E) 10.5 and 13.5. This progenitor pool expands in the ventricular zone (VZ) through symmetric cell division until E10.5, when a gradual switch to asymmetric cell division occurs, regulated by Notch1 (Lutolf et al., 2002) and its interactor (Masserdotti et al., 2010), the Zn-finger TF Zfp423 (Alcaraz et al., 2006; Warming et al., 2006; Croci et al., submitted). Zfp423 was recently implicated in Joubert syndrome and cerebellar vermis hypoplasia (Chaki et al., 2012). Allelic mutations of Zfp423 produce distinct alterations in PC development (Croci et al., submitted). PCs arise from a pool of progenitors positive for the basic-helix-loop-helix transcription factors (TFs) neurogenin (Ngn) 1 and 2 (Zordan et al., 2008; Lundell et al., 2009). Ngn2 regulates cell cycle progression and dendritic arbor generation in PC precursors (Florio et al., 2012). PCs also express HLH transcription factors of the Olf/EBF family. In Ebf2 -/- mutants, PC migration and survival are affected (Croci et al., 2006). Neonatal PC death is due to local downregulation of Igf1 gene expression (Croci et al., 2011). Finally, EBF2 regulates cortical patterning in the adult cerebellum, regulating its subdivision into alternate parasagittal stripes of distinct PC subtypes. Indeed, EBF2 is required to repress the zebrin II+ phenotype in postnatal PCs (Croci et al., 2006; Chung et al., 2008)

ZFP423, a transcription factor implicated in Joubert Syndrome and Cerebellar Vermis Hypoplasia, orchestrates the pace and mode of cerebellar neurogenesis

Neurogenesis is a tightly regulated process, both in the embryonic and in the adult brain. Its success depends on the ability of a germinative epithelium to establish the appropriate balance between maintaining an undifferentiated progenitor pool and giving birth to sequential generations of neurons and glia. The Zfp423 gene encodes a 30 Zn-finger transcription factor (TF) which interacts with the SMAD1- SMAD4 complex (BMP signaling), Notch intracellular domain, retinoic acid receptors and Collier/Olf-1/EBF TFs. This gene has been previosly implicated in cerebellar development. Mutations in the human ortholog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), and/ or exhibiting other signs of ciliopathy outside the central nervous system. We have been analyzing two mouse mutant lines carrying allelic in-frame deletions of Zfp423. One of them lacks Zn-finger domains 9-20 (Δ9-20), implicated in BMP and Notch signal transduction, while the other lacks a C-terminal domain (Δ28-30). Both mutants exhibit cerebellar malformations and severe ataxia. However, our results indicate that the two protein domains play sharply distinct roles in the context of cerebellar neurogenesis. In Zfp423Δ9-20/Δ9-20 mutants, GABAergic Purkinje cell (PC) neurogenesis is impaired and the PC progenitor pool in the ventricular zone is precociously depleted. Conversely, Zfp423Δ28-30/Δ28-30 mutants display a selective impairment in the development of glutamatergic cerebellar neurons

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging

Visualization and Identification of IL-7 Producing Cells in Reporter Mice

Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging