Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

doi: 10.1007/978-1-0716-2201-8_19

Figure Lengend Snippet: (A) Results of an egg hatching assay using the classical amx allele over a molecularly defined deficiency obtained from the Bloomington Drosophila Stock Center (BDSC). None of the eggs laid by the amx1/Df(1)Exel9094 mothers hatch, while a significant portion of this can be rescued by introduction of a wild-type amx genomic rescue construct (+amx). The maximum rescue expected in this experiment is 75% because Df(1)Exel9094 removes a nearby essential gene [Df(1)9094/Y males are lethal regardless of amx function]. Humanized amx genomic rescue transgene (+TM2D3) has about 50% activity of the fly amx genomic rescue transgene and can rescue the egg hatching defect of amx1/Df(1)Exel9094 progenies. In contrast, the humanized amx genomic rescue construct with the late-onset Alzheimer’s disease (LOAD) associated p.P155L variant (+TM2D3p.P155L) lacks this activity. (B) Results of an egg hatching assay using the newly generated amx null allele via CRISpR. The results obtained from amxΔ are similar to the results using amx1/Df(1)Exel9094, further confirming the results using the classic allele. (C-F) Representative images of the nervous system of embryos that are mutant for amx rescued by different genomic rescue constructs. Neurons are visualized by immunofluorescence staining against Elav, a pan-neuronal nuclear marker. (C) Embryos from amx1/Df(1)Exel9094 mothers show a strong neurogenic phenotype, which is a classic sign of Notch signaling defect during embryonic neurogenesis. (D) A wild-type amx genomic rescue transgene can fully rescue this defect. (E) A humanized amx genomic transgene expressing the reference human TM2D3 can also rescue this defect in a subset of animals. (F) A humanized amx genomic transgene expressing human TM2D3 with the p.P155L variant cannot rescue the neurogenic defect, indicating this missense variant is deleterious to a Notch signaling related function of this gene in vivo. Similar results have been obtained using the amxΔ allele (data not shown). Scale bars = 100 μm. Images were adopted from (33).

Article Snippet: Reagents: 10 mM ATP (NEB, P0756S) 10X T4 ligation buffer (NEB, B0202S) Antarctic Phosphatase (NEB, M0289S) BbsI (NEB, R0539S) BsaI-HFv2 (NEB, R3733S) CutSmart buffer (NEB, B7204S) ddH 2 O (double distilled autoclaved water) DH5α competent bacteria LB media Max Efficiency Stbl2 competent bacteria (Thermo Fisher # 10268019) Miniprep or Midiprep kit (e.g. QIAprep Spin Miniprep Kit or Plasmid Midi Kit, QIAGEN, 28104 or 12143) NheI (NEB, R0131) P[acman] clone CH322-146A15 (BACPAC resources, CH322-146A15) pattB (DGRC, 1420) pBH vector ( 83 ) PCR Purification Kit (e.g. QIAquick PCR Purification Kit, QIAGEN, 27104) pCFD3-dU6:3gRNA plasmid (Addgene #49410) Q5® Site-Directed Mutagenesis Kit (NEB, E0554S) T4 DNA ligase (NEB, M0202S) T4 Polynucleotide Kinase (NEB, M0201S) TM2D3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_078474","term_id":"1519241918","term_text":"NM_078474"}} NM_078474 ) Human Tagged ORF Clone (Origene, RC203014) XhoI (NEB, R0146S) XbaI (NEB, R0145S) yellow[wing2+] template vector ( 84 ) Equipment and supplies: 0.2 ml PCR tubes (e.g. Eppendorf) 1.5 ml centrifuge tubes (e.g. Eppendorf) 15 ml centrifuge tubes (e.g. Corning) Centrifuges (e.g.

Techniques: Construct, Activity Assay, Variant Assay, Generated, CRISPR, Mutagenesis, Immunofluorescence, Staining, Marker, Expressing, In Vivo

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

doi: 10.1007/978-1-0716-2201-8_19

Figure Lengend Snippet: (A) Schematic diagram of the amx locus on the Drosophila X-chromosome. The coding region of genes are shown as orange boxes and the untranslated regions (UTR) are shown in grey. Two primers (Oligo 1 and 2) were designed to amplify a ~3.3kb region that contains the amx gene and neighboring sequences. (B) Construction of the amx[+]-attB genomic rescue construct by subcloning the PCR amplicon from (A) into pattB using XhoI and XbaI digestion and ligation. pattB contains a mini-white+ cassette (red box) as a dominant visual marker that can be used to track the transgenesis event in a white− mutant background. It also carries an Ampicillin resistance (AmpR) cassette (green box) for selection in bacteria. (C) Construction of a humanized amx genomic rescue construct (TM2D3[+]-attB) based on amx[+]-attB and two intermediate vectors. Human reference TM2D3 coding sequence was PCR amplified from a cDNA plasmid and integrated into a plasmid in which the amx coding sequence was replaced by a spacer sequence flanking two BsaI recognition sites (Intermediate Vector 2). A construct in which the upstream region of the amx genomic rescue amplicon with a BsaI site was cloned into pattB (Intermediate Vector 1) as a precursor of Intermediate Vector 2. Golden Gate cloning with BsaI and overhangs ①/② allows swapping of the spacer sequence in Intermediate Vector 2 with human TM2D3 coding sequence to generate the humanized genomic rescue construct. (D) Generation of the humanized amx genomic rescue construct carrying the variant of interest (TM2D3[P155L]-attB) through site-directed mutagenesis. The variant of interest is shown using an asterisk.

Article Snippet: Reagents: 10 mM ATP (NEB, P0756S) 10X T4 ligation buffer (NEB, B0202S) Antarctic Phosphatase (NEB, M0289S) BbsI (NEB, R0539S) BsaI-HFv2 (NEB, R3733S) CutSmart buffer (NEB, B7204S) ddH 2 O (double distilled autoclaved water) DH5α competent bacteria LB media Max Efficiency Stbl2 competent bacteria (Thermo Fisher # 10268019) Miniprep or Midiprep kit (e.g. QIAprep Spin Miniprep Kit or Plasmid Midi Kit, QIAGEN, 28104 or 12143) NheI (NEB, R0131) P[acman] clone CH322-146A15 (BACPAC resources, CH322-146A15) pattB (DGRC, 1420) pBH vector ( 83 ) PCR Purification Kit (e.g. QIAquick PCR Purification Kit, QIAGEN, 27104) pCFD3-dU6:3gRNA plasmid (Addgene #49410) Q5® Site-Directed Mutagenesis Kit (NEB, E0554S) T4 DNA ligase (NEB, M0202S) T4 Polynucleotide Kinase (NEB, M0201S) TM2D3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_078474","term_id":"1519241918","term_text":"NM_078474"}} NM_078474 ) Human Tagged ORF Clone (Origene, RC203014) XhoI (NEB, R0146S) XbaI (NEB, R0145S) yellow[wing2+] template vector ( 84 ) Equipment and supplies: 0.2 ml PCR tubes (e.g. Eppendorf) 1.5 ml centrifuge tubes (e.g. Eppendorf) 15 ml centrifuge tubes (e.g. Corning) Centrifuges (e.g.

Techniques: Construct, Subcloning, Amplification, Ligation, Marker, Mutagenesis, Selection, Sequencing, Plasmid Preparation, Clone Assay, Variant Assay