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Mouse Pdlim1 Conditional Knockout Project (CRISPR/Cas9)

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https://www.alphaknockout.com Mouse Pdlim1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Pdlim1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Pdlim1 gene (NCBI Reference Sequence: NM_016861 ; Ensembl: ENSMUSG00000055044 ) is located on Mouse chromosome 19. 7 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 7 (Transcript: ENSMUST00000068439). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Pdlim1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP24-352A5 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a gene trap allele exhibit enhanced platelet response to GPVI agonists and thrombosis. Exon 2 starts from about 9.89% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 19284 bp, and the size of intron 2 for 3'-loxP site insertion: 2206 bp. The size of effective cKO region: ~652 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 7 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Pdlim1 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7152bp) | A(27.39% 1959) | C(22.04% 1576) | T(28.22% 2018) | G(22.36% 1599) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 7 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr19 - 40252312 40255311 3000 browser details YourSeq 210 2642 2991 3000 89.5% chr12 - 70005272 70005621 350 browser details YourSeq 209 2640 2982 3000 88.2% chr11 - 54078387 54078749 363 browser details YourSeq 209 2641 2976 3000 90.8% chr1 + 91140449 91140803 355 browser details YourSeq 207 2640 2991 3000 91.9% chr1 + 118696724 118697085 362 browser details YourSeq 204 2642 2992 3000 89.4% chr8 - 85707118 85707484 367 browser details YourSeq 203 2642 3000 3000 85.9% chr1 - 183237369 183237696 328 browser details YourSeq 200 2642 3000 3000 89.5% chr4 + 46217186 46217550 365 browser details YourSeq 200 2646 2965 3000 85.3% chr17 + 43927872 43928180 309 browser details YourSeq 199 2642 2972 3000 85.8% chr13 + 91267135 91267406 272 browser details YourSeq 199 2642 2995 3000 85.8% chr11 + 104862662 104863011 350 browser details YourSeq 197 2652 3000 3000 87.4% chr16 + 37803679 37804024 346 browser details YourSeq 197 2220 2992 3000 82.0% chr1 + 63521947 63522675 729 browser details YourSeq 196 2641 3000 3000 86.9% chr9 - 77861022 77861365 344 browser details YourSeq 196 2642 2986 3000 87.2% chr5 - 99216770 99217173 404 browser details YourSeq 195 2683 2983 3000 86.7% chr7 + 79061802 79062070 269 browser details YourSeq 195 2684 3000 3000 87.1% chr10 + 83762906 83763184 279 browser details YourSeq 194 2644 3000 3000 86.5% chr2 + 22247330 22247689 360 browser details YourSeq 194 2650 2990 3000 90.2% chr18 + 74055498 74055916 419 browser details YourSeq 194 2642 2985 3000 83.3% chr11 + 50169468 50169768 301 Note: The 3000 bp section upstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr19 - 40248660 40251659 3000 browser details YourSeq 199 898 1199 3000 88.4% chr7 + 43629613 43629928 316 browser details YourSeq 185 911 1210 3000 86.7% chr16 - 4734222 4734542 321 browser details YourSeq 162 916 1216 3000 86.3% chr5 - 132824722 132825039 318 browser details YourSeq 153 898 1211 3000 81.2% chr12 - 106562317 106562627 311 browser details YourSeq 149 811 1211 3000 78.1% chr3 + 65167146 65167488 343 browser details YourSeq 149 932 1191 3000 89.6% chr16 + 24082330 24082611 282 browser details YourSeq 147 937 1191 3000 89.8% chr8 - 86612121 86612394 274 browser details YourSeq 145 909 1210 3000 90.1% chr19 + 25042486 25042793 308 browser details YourSeq 144 908 1211 3000 88.4% chr7 - 143530206 143530514 309 browser details YourSeq 141 882 1184 3000 88.5% chr4 + 154874446 154874765 320 browser details YourSeq 140 910 1202 3000 85.2% chr2 + 10988803 10989118 316 browser details YourSeq 137 911 1186 3000 90.3% chr6 - 51776102 51776403 302 browser details YourSeq 135 933 1162 3000 87.4% chr11 - 58133509 58133747 239 browser details YourSeq 135 875 1201 3000 84.7% chr11 - 24916522 24916866 345 browser details YourSeq 135 908 1210 3000 88.8% chr10 - 3327772 3328094 323 browser details YourSeq 133 880 1201 3000 85.5% chr13 + 96759640 96759975 336 browser details YourSeq 132 811 1162 3000 77.3% chr4 - 109195536 109195801 266 browser details YourSeq 132 909 1164 3000 85.6% chr12 - 25089229 25089479 251 browser details YourSeq 132 937 1203 3000 88.6% chr5 + 67074594 67074871 278 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Pdlim1 PDZ and LIM domain 1 (elfin) [ Mus musculus (house mouse) ] Gene ID: 54132, updated on 10-Oct-2019 Gene summary Official Symbol Pdlim1 provided by MGI Official Full Name PDZ and LIM domain 1 (elfin) provided by MGI Primary source MGI:MGI:1860611 See related Ensembl:ENSMUSG00000055044 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as CLP36; Clim1; mClim1 Expression Broad expression in large intestine adult (RPKM 123.7), placenta adult (RPKM 92.4) and 19 other tissues See more Orthologs human all Genomic context Location: 19; 19 C3 See Pdlim1 in Genome Data Viewer Exon count: 7 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 19 NC_000085.6 (40222239..40271616, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 19 NC_000085.5 (40296729..40346106, complement) Chromosome 19 - NC_000085.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Pdlim1 ENSMUSG00000055044 Description PDZ and LIM domain 1 (elfin) [Source:MGI Symbol;Acc:MGI:1860611] Gene Synonyms CLP36, mClim1 Location Chromosome 19: 40,221,173-40,271,842 reverse strand. GRCm38:CM001012.2 About this gene This gene has 5 transcripts (splice variants), 219 orthologues, 8 paralogues, is a member of 1 Ensembl protein family and is associated with 9 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Pdlim1-201 ENSMUST00000068439.12 2571 327aa ENSMUSP00000064545.5 Protein coding CCDS37977 O70400 TSL:1 GENCODE basic APPRIS P1 Pdlim1-202 ENSMUST00000182432.1 692 198aa ENSMUSP00000138383.1 Protein coding - S4R1V0 CDS 3' incomplete TSL:3 Pdlim1-204 ENSMUST00000182636.1 2835 No protein - Retained intron - - TSL:1 Pdlim1-203 ENSMUST00000182540.1 715 No protein - Retained intron - - TSL:2 Pdlim1-205 ENSMUST00000182813.1 666 No protein - Retained intron - - TSL:2 70.67 kb Forward strand Genes Gm16470-201 >processed pseudogene (Comprehensive set... Contigs AC170187.2 > Genes (Comprehensive set... < Pdlim1-201protein coding < Pdlim1-203retained intron < Pdlim1-204retained intron < Pdlim1-205retained intron < Pdlim1-202protein coding Regulatory Build Reverse strand 70.67 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding pseudogene processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000068439 < Pdlim1-201protein coding Reverse strand 50.44 kb ENSMUSP00000064... Superfamily PDZ superfamily SSF57716 SMART PDZ domain Zasp-like motif Zinc finger, LIM-type Pfam PDZ domain Domain of unknown function DUF4749 Zinc finger, LIM-type PROSITE profiles PDZ domain Zinc finger, LIM-type PROSITE patterns Zinc finger, LIM-type PANTHER PTHR24214 PDZ and LIM domain protein 1 Gene3D 2.30.42.10 2.10.110.10 CDD cd00992 cd09448 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 280 327 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
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  • Identifying Dynamic Protein and RNA Proximity Interaction Networks of Actinin Reveals RNA

    Identifying Dynamic Protein and RNA Proximity Interaction Networks of Actinin Reveals RNA

    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license. 1 Title: 2 Identifying dynamic protein and RNA proximity interaction networks of actinin reveals RNA- 3 binding and metabolic regulatory mechanisms 4 5 Feria A. Ladha M.S.1, Ketan Thakar Ph.D.2*, Anthony M. Pettinato B.S.1*, Nicholas Legere 2 2 1 1 2 6 B.S. , Rachel Cohn M.S. , Robert Romano B.S. , Emily Meredith B.S. , Yu-Sheng Chen Ph.D. , 7 and J. Travis Hinson M.D.1,2,3 8 Affiliations: 9 1University of Connecticut Health Center, Farmington, CT 06030, USA 10 2The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA 11 3Cardiology Center, UConn Health, Farmington, CT 06030, USA 12 *These authors contributed equally 13 14 Lead Contact: Correspondence: [email protected] 15 16 17 18 19 20 21 22 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.18.994004; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available underConfidential aCC-BY-NC-ND Manuscript 4.0 International license. 1 Abstract 2 Actinins are actin cross-linkers that are expressed in nearly all cells and harbor mutations in 3 heritable diseases.

  • Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing

    www.nature.com/scientificreports OPEN Transcriptomic Profling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq Received: 11 January 2019 Shahensha Shaik1, Elizabeth C. Martin2, Daniel J. Hayes3, Jefrey M. Gimble4 & Accepted: 25 July 2019 Ram V. Devireddy1 Published: xx xx xxxx Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be diferentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to diferentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC diferentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profle of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the diferentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-afliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from diferentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF- β, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways.