Journal: Cell reports

Article Title: Distinct Mechanisms of Over-Representation of Landmarks and Rewards in the Hippocampus

doi: 10.1016/j.celrep.2020.107864

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse Thy1.2 promoter , Feng et al., 2000 , RRID: Addgene_20736.

Techniques: Recombinant, Software, Imaging, Modification

Journal: Nature communications

Article Title: Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.

doi: 10.1038/s41467-024-55604-0

Figure Lengend Snippet: Fig. 3 | Expression of pegRNA with a POL II-promoter enhances prime editing delivery. a Structure of post-splicing ipegRNA and epegRNA.Created in BioRender. Ohlmann, T. (2024) https://BioRender.com/g43z718. b Efficacy of PE measured in SWYS cells transduced with GAG-PEV4-VLPs produced from cells expressing intronic-pegRNAs. Production was performed with addition of the Csy4 protein or its non-cleaving mutant Csy4H29A. (n = 9) c Comparison of GAG-PEV4 and GAG-PE- V7 for the production of ipeg-loaded VLPs. (n = 9). d Impact of ipegRNA on VLP-

Article Snippet: Plasmids coding for PE2 (#132775), PEmax (#174820) and Csy4 (55196) were obtained from Addgene.

Techniques: Expressing, Transduction, Produced, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: TESOGENASE, An Engineered Nuclease Editor for Enhanced Targeted Genome Integration

doi: 10.1101/2023.08.28.553855

Figure Lengend Snippet: A, Schematic diagram of various Cas9-homologous recombination protein fusion constructs (enGagers) in all-in-one (AIO) plasmid format modified from Addgene plasmid #42230. Two nuclear localization signals were added to the N’ and C’-termini of the Cas9 protein. RecA is the bacteria homologous DNA repair protein and Rad51(AE)/Rad51(SEAD) are two mutant variants from eukaryotes. Brex is a 36 amino acid peptide reported to recruit Rad51 in mammalian cells. B, Schematic diagram of Knock in strategy of a 2Kb (left) and 4Kb (right) cssDNA donor template for RAB11A locus. C, representative FACS profiles with gating strategy showing % of GFP transgene cassette Knock in on RAB11 locus at day 3 post electroporation for various enGagers listed in A. D, Quantification of 2Kb GFP transgene cassette Knock in fold change of various enGagers as compared to Cas9 WT at day 3 (left), 8 (middle) and 14 (right) post electroporation. E, representative FACS profiles with gating strategy showing % of 4Kb GFP transgene cassette Knock in on RAB11 locus at day 3 post electroporation for various enGagers listed in A. F, Quantification of 4Kb GFP transgene cassette Knock in fold change of various enGagers as compared to Cas9 WT at day 3 (left), 7 (right) post electroporation. Note that Brex enGager does not enhance knock in efficiency. Rad51 mutants and RecA enGagers increase both small and large transgene cassette knock in by 1.57 to 3.04-fold. RecA enGager outperforms among the enGagers tested. Bars represent mean ± SD from 3 biological replicates.

Article Snippet: For all-in-one (AIO) plasmid construct design for various enGagers (Table 1), ssDNA binding protein and peptide sequences were synthesized and assembled into an AIO plasmid modified from Addgene plasmid #42230 by golden gate cloning.

Techniques: Homologous Recombination, Construct, Plasmid Preparation, Modification, Bacteria, Mutagenesis, Knock-In, Electroporation

Journal: bioRxiv

Article Title: TESOGENASE, An Engineered Nuclease Editor for Enhanced Targeted Genome Integration

doi: 10.1101/2023.08.28.553855

Figure Lengend Snippet: A, Schematic diagram of various Cas9-ssDNA binding motifs fusion constructs (enGagers) in all-in-one (AIO) plasmid format modified from Addgene plasmid #42230. Two nuclear localization signals were added to the N’ and C’-termini of the Cas9 protein. Cas9-RecA fusion construct was used as a positive control. FECO, WECO and YECO are 20 amino acid sequences previously identified as ssDNA binding motifs in various bacteria species of RecA, FECO3X, WECO3X and YECO3X are 3 tandem copies of the 20 aa peptides separated by multi-GS peptide linkers. B, Schematic diagram of Knock in strategy of a 2Kb cssDNA donor template for RAB11A locus. C, representative FACS profiles with gating strategy showing % of GFP transgene cassette Knock in on RAB11 locus at day 7 post electroporation for various small enGagers listed in A. D, Quantification of 2Kb GFP transgene cassette Knock in fold change of various enGagers as compared to Cas9 WT at day 7 post electroporation. Note that Cas9-FECO fusion performs similarly with Cas9-RecA fusion in cssDNA mediated transgene integration (1.59-vs 1.58-fold). EnGagers with 3X tandem ssDNA binding peptides do not further enhance knock in efficiency does not enhance knock in efficiency. Bars represent mean ± SD from 3 biological replicates.

Article Snippet: For all-in-one (AIO) plasmid construct design for various enGagers (Table 1), ssDNA binding protein and peptide sequences were synthesized and assembled into an AIO plasmid modified from Addgene plasmid #42230 by golden gate cloning.

Techniques: Binding Assay, Construct, Plasmid Preparation, Modification, Positive Control, Bacteria, Knock-In, Electroporation

Journal: bioRxiv

Article Title: TESOGENASE, An Engineered Nuclease Editor for Enhanced Targeted Genome Integration

doi: 10.1101/2023.08.28.553855

Figure Lengend Snippet: A, Schematic diagram of various Cas9-ssDNA binding protein and peptide fusion constructs (enGagers) in all-in-one (AIO) plasmid format modified from Addgene plasmid #42230. Two nuclear localization signals were added to the N’ and C’-termini of the Cas9 protein. Cas9-RecA and Cas9-FECO fusion constructs were used as positive controls. The fusion protein or peptides include DrRecA, 20 aa motif identified from DrRecA, SSAP, Lambda Red, RecT, RadA and RadB from Archaea. B, Schematic diagram of Knock in strategy of a 2Kb cssDNA donor template for RAB11A locus. C, Quantification of 2Kb GFP transgene cassette Knock in fold change of various enGagers as compared to Cas9 WT at day 7 post electroporation. Note that Cas9-DrRecA and Cas9-DrRecA20AA fusion has the highest performance in knock in with 2.17- and 2.43-fold as compared to Cas9 WT, respectively. D. Quantification of cell viability day 7 post electroporation. Bars represents mean ± SD from 3 biological replicates. E. Amino acid sequence alignment of 20AA of multiple E.coli RecA mutant variants and RecA from archaea and mammalian organism. Dr: Deinococcus radiodurans ; Ec: Escherichia coli; Sc: Saccharomyces cerevisiae; Hs: Homo sapiens; Pf: P. furiosus; Sso: S. solfatarcus .

Article Snippet: For all-in-one (AIO) plasmid construct design for various enGagers (Table 1), ssDNA binding protein and peptide sequences were synthesized and assembled into an AIO plasmid modified from Addgene plasmid #42230 by golden gate cloning.

Techniques: Binding Assay, Construct, Plasmid Preparation, Modification, Knock-In, Electroporation, Sequencing, Mutagenesis

Journal: Cell Proliferation

Article Title: Transcription factor PBX4 regulates limb development and haematopoiesis in mice

doi: 10.1111/cpr.13580

Figure Lengend Snippet: PBX4 is specifically expressed in mouse testis. (A) Schematic diagrams of Pbx4 gene structure and PBX4 protein domains. Blue and white rectangles indicate protein‐coded exons and UTR regions, respectively. (B) Left panel: detection of Pbx4 expression by RT‐PCRs in various tissues of adult mice. Right panel: quantitative RT‐PCR results of Pbx4 expression in different testicular cells. eST, elongating spermatids; pacSC, pachytene spermatocytes; plpSC, preleptotene spermatocytes; rST, round spermatids; Sertoli, Sertoli cells; SG‐A, type A spermatogonia; SG‐B, type B spermatogonia. The expression levels are normalised by that of SG‐A, n ≥ 3. (C) Schematic diagram of PBX4‐FLAG knockin (KI) strategy. LHA: left homologous arm; RHA: right homologous arm. (D) Western blot analyses of PBX4 in multiple mouse organs using α‐FLAG. * labels the absence of ACTB signal in heart and rectus femoris samples due to low expression levels. Equal amounts of total protein in all samples were loaded based on quantification using the BCA kit. (E) Immunohistochemical (IHC) staining of PBX4‐FLAG in testicular sections using α‐FLAG. pacSC, pachytene spermatocytes; rST, round spermatids. Scale bar, 20 μm.

Article Snippet: For PBX4‐FLAG KI mice, donor DNA fragment containing the left and right homologous arms flanking the 3 × FLAG coding sequence was cloned into a plasmid modified from a knockin targeting construct (#59721, addgene) and amplified in bacteria.

Techniques: Expressing, Quantitative RT-PCR, Knock-In, Western Blot, Immunohistochemical staining, Immunohistochemistry

Journal: Cell Proliferation

Article Title: Transcription factor PBX4 regulates limb development and haematopoiesis in mice

doi: 10.1111/cpr.13580

Figure Lengend Snippet: Pbx4 knockout (KO) does not affect mouse spermatogenesis. (A) Schematic illustration of the predicted effects of Pbx4 KO on the proteins encoded by transcript variant 1 (v1) and 2 (v2). Gene KO using sgRNA2‐1 and sgRNA2‐2 resulted in two mutant alleles KO‐2‐d4 and KO‐2‐d8, which were confirmed by sequencing. The corresponding predicted truncated proteins were labelled by the names of these two alleles. (B) Genotyping results of WT, HET (heterozygous deletion) and KO (homozygous deletion) mice by PCRs with 120F and 120R primers. (C) Validation of KO of PBX4 by Western blotting with α‐PBX4. The PBX4‐FLAG knockin (KI) mouse was used as a control for the PBX4 signal on Western blot. The original full image is shown as Figure . (D) The morphology of WT and KO mice and their testes and epididymis. (E) Comparison of litter sizes of WT and KO mice. (F) Haematoxylin and eosin staining of testicular and epididymis sections in WT and KO mice. Scale bar, 50 μm.

Article Snippet: For PBX4‐FLAG KI mice, donor DNA fragment containing the left and right homologous arms flanking the 3 × FLAG coding sequence was cloned into a plasmid modified from a knockin targeting construct (#59721, addgene) and amplified in bacteria.

Techniques: Knock-Out, Variant Assay, Mutagenesis, Sequencing, Biomarker Discovery, Western Blot, Knock-In, Control, Comparison, Staining