Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in Caenorhabditis elegans.

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the flippase (FLP) and cyclization recombination (Cre) enzymes has proved particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes, and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for green fluorescent fusion proteins based on FLP-mediated recombination. Using 2 stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify transcriptomes in a C. elegans tissue of interest. We have adapted RNA polymerase DamID for the FLP toolbox and by focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by RNA polymerase DamID are known to be expressed in this tissue. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

A Flp-dependent G-CaMP9a transgenic mouse for neuronal imaging in vivo.

Genetically encoded calcium indicators (GECIs) are widely used to measure calcium transients in neuronal somata and processes, and their use enables the determination of action potential temporal series in a large population of neurons. Here, we generate a transgenic mouse line expressing a highly sensitive green GECI, G-CaMP9a, in a Flp-dependent manner in excitatory and inhibitory neuronal subpopulations downstream of a strong CAG promoter. Combining this reporter mouse with viral or mouse genetic Flp delivery methods produces a robust and stable G-CaMP9a expression in defined neuronal populations without detectable detrimental effects. In vivo two-photon imaging reveals spontaneous and sensory-evoked calcium transients in excitatory and inhibitory ensembles with cellular resolution. Our results show that this reporter line allows long-term, cell-type-specific investigation of neuronal activity with enhanced resolution in defined populations and facilitates dissecting complex dynamics of neural networks in vivo.

The wmN1 Enhancer Region of the Mouse Myelin Proteolipid Protein Gene (mPlp1) is Indispensable for Expression of an mPlp1-lacZ Transgene in Both the CNS and PNS.

The myelin proteolipid protein gene (PLP1) encodes the most abundant protein in CNS myelin. Expression of the gene must be strictly regulated, as evidenced by human X-linked leukodystrophies resulting from variations in PLP1 copy number, including elevated dosages as well as deletions. Recently, we showed that the wmN1 region in human PLP1 (hPLP1) intron 1 is required to promote high levels of an hPLP1-lacZ transgene in mice, using a Cre-lox approach. The current study tests whether loss of the wmN1 region from a related transgene containing mouse Plp1 (mPlp1) DNA produces similar results. In addition, we investigated the effects of loss of another region (ASE) in mPlp1 intron 1. Previous studies have shown that the ASE is required to promote high levels of mPlp1-lacZ expression by transfection analysis, but had no effect when removed from the native gene in mouse. Whether this is due to compensation by another regulatory element in mPlp1 that was not included in the mPlp1-lacZ constructs, or to differences in methodology, is unclear. Two transgenic mouse lines were generated that harbor mPLP(+)Z/FL. The parental transgene utilizes mPlp1 sequences (proximal 2.3 kb of 5'-flanking DNA to the first 37 bp of exon 2) to drive expression of a lacZ reporter cassette. Here we demonstrate that mPLP(+)Z/FL is expressed in oligodendrocytes, oligodendrocyte precursor cells, olfactory ensheathing cells and neurons in brain, and Schwann cells in sciatic nerve. Loss of the wmN1 region from the parental transgene abolished expression, whereas removal of the ASE had no effect.

Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system.

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off-target sites revealed no mutation event. Moreover, our construct contained a heat-shock inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.

Assembling Multi-subunit Complexes Using Mammalian Expression.

In this chapter conventional and emerging new technologies for the production of complex biologics in mammalian expression systems are summarized. The essential features of the most relevant methods to generate stable production cell lines for the expression of recombinant multi-protein complexes are described. Especially the promising multiple targeted integration strategy by Flp or CRISPR/Cas9 mediated recombination and their future impact on multi-protein expression are highlighted.

Tools for advanced and targeted genetic manipulation of the ß-lactam antibiotic producer Acremonium chrysogenum.

Acremonium chrysogenum is the major producer of the ß-lactam antibiotic cephalosporin C and therefore of great importance for the pharmaceutical industry. However, this filamentous fungus is known to reproduce solely by asexual means, shows only sporadic conidiospore production, and has gradual fragmentation of the vegetative mycelium into arthrospores. Due to these peculiar growth characteristics and life style, strain improvement by recombinant technologies is much more challenging than for other biotechnologically relevant fungi. Here, we describe several molecular tools for genetic engineering of A. chrysogenum, including a ΔAcku70 deletion strain for homologous recombination. No physiological or morphological changes occurred due to deletion of the ku70 gene or integration of the nat1 cassette in this recipient strain. We also used a xylose-inducible promoter from Sordaria macrospora (Smxyl) to demonstrate induction of the gfp reporter gene in A. chrysogenum. The Smxyl promoter was used for construction of a vector molecule to develop a one-step FLP/FRT recombination system in A. chrysogenum. This system was then used in the ΔAcku70 deletion strain to construct a marker-free recipient strain for targeted DNA insertion into genomic DNA. The applicability of our tools was demonstrated by construction of a marker-free transgenic strain, lacking any foreign genes.