paci  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: RNA aptamer inhibitors of a restriction endonuclease

doi: 10.1093/nar/gkv702

Figure Lengend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

Article Snippet: Protein expression and purification Commercial REases and purified preparations of BamHI (E111A mutant), KpnI and PacI for in vitro selection and binding assays were obtained from New England Biolabs.

Techniques: In Vitro, Selection

Journal: Nucleic Acids Research

Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

doi: 10.1093/nar/gkl635

Figure Lengend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

Article Snippet: A total of 5 μg plasmid DNA of the constructed vectors were digested with 40 U PacI (New England Biolabs) overnight at 37°C in a total volume of 200 μl.

Techniques: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

Journal: Nucleic Acids Research

Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

doi: 10.1093/nar/gkl635

Figure Lengend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

Article Snippet: A total of 5 μg plasmid DNA of the constructed vectors were digested with 40 U PacI (New England Biolabs) overnight at 37°C in a total volume of 200 μl.

Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

Journal: Tropical Medicine and Infectious Disease

Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

doi: 10.3390/tropicalmed2030037

Figure Lengend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

Article Snippet: The amplified PCR product was digested with XmaI and PacI (New England Biolabs, Ipswich, MA, USA) and then ligated to pSPBN previously digested with XmaI and PacI .

Techniques: Recombinant

Journal: bioRxiv

Article Title: Parallel functional testing identifies enhancers active in early postnatal mouse brain

doi: 10.1101/2021.01.15.426772

Figure Lengend Snippet: Vector map of pscAAV-Hsp68-EGFP. Vector map of the self-complementary previral vector used for cloning. Inset (bottom) shows multiple cloning site for Gibson assembly and amplicon PCR primer locations.

Article Snippet: We next linearized the vector pscAAV-Hsp68-EGFP ( ) using PacI (NEB #R0547L) and AscI (NEB #R0558L).

Techniques: Plasmid Preparation, Clone Assay, Amplification, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Parallel functional testing identifies enhancers active in early postnatal mouse brain

doi: 10.1101/2021.01.15.426772

Figure Lengend Snippet: In vivo parallelized functional enhancer reporter screen. ( A ) Schematic of in vivo parallelized functional enhancer reporter assay. The test library was generated using the previral vector pscAAV-Hsp68-EGFP, which contained a multiple cloning site (light grey) between the EGFP reporter and polyadenylation site (PAS). Purified PCR amplicons of test amplicons were cloned into the vector using Gibson assembly. The previral library was packaged into AAV9(2YF), and the viral library delivered to the brain via ventricular injection at postnatal day (P)0. Brains were collected at P7. ( B ) Correlation of genomic DNA (“DNA”) representation across biological replicates (L1-L4) and the previral plasmid library. Data is shown as log 2 (proportion) per amplicon. Pearson correlation is shown for each pairwise comparison. ( C ) Correlation of cDNA (“RNA”) representation across biological replicates (L1-L4) and the technical replicate L4.2. Data is shown as log 2 (proportion) per amplicon. Pearson correlation is shown for each pairwise comparison. ( D ) ( left ) Correlation of mean RNA/DNA ratio in the assay of all amplicons detected in the previral library (n = 408). Dashed line represents RNA/DNA best-fit line of the data (n = 247). Amplicons are colored by whether they were found significant using the multiple linear model. ( right ) Amplicons tested by the multiple linear model (n = 321), with those with significant (P norm

Article Snippet: We next linearized the vector pscAAV-Hsp68-EGFP ( ) using PacI (NEB #R0547L) and AscI (NEB #R0558L).

Techniques: In Vivo, Functional Assay, Reporter Assay, Generated, Plasmid Preparation, Clone Assay, Purification, Polymerase Chain Reaction, Injection, Amplification

Journal: bioRxiv

Article Title: Parallel functional testing identifies enhancers active in early postnatal mouse brain

doi: 10.1101/2021.01.15.426772

Figure Lengend Snippet: Functional validation of STARR-seq screen. ( A-C ) Enhancers retain cell-type specific function in the STARR-seq vector. ( A ) Representative image of a coronal section of a P7 mouse brain injected at P0 with a virus mixture consisting of an AAV containing the STARR-seq vector carrying the inhibitory interneuron enhancer mDlx (scAAV9-Hsp68-EGFP-mDlx) and an injection control AAV containing an expression vector for mRuby3 under the control of CAG, a general mammalian promoter. EGFP expression was visualized via IHC using an anti-GFP antibody, while mRuby3 expression was visualized using native fluorescence. ( B ) Close up of boxed regions in A showing morphology of EGFP-expressing cells in the cortex. ( C ) Sections from P7 mouse cortex transduced with mDlx -driven STARR-seq reporter vector and mRuby3 injection control at P0, counterstained with an antibody for Lhx6 , a transcription factor active in deep cortical layer interneurons. EGFP-expressing cells with Lhx6 + Nuclei are indicated with arrows. ( D-E ) Validation of positive and negative hits from in vivo STARR-seq screen. ( D ) The enhancer candidate amplicon #161, a region overlapping a DNaseI hypersensitive site in fetal human brain and a copy number variant region near SCN2A , an autism- and epilepsy-associated gene, which displayed enhancer activity in the STARR-seq screen, was packaged individually into an AAV reporter vector and transduced along with an injection control (CAG-mRuby3) by intracranial injection in P0 mice. A representative image of a coronal section of a transduced P7 brain stained with an anti-GFP antibody is shown (left panel). Close up of the boxed region is shown in the panels on the right (from left to right: Red channel, mRuby3 injection control; Green channel, EGFP expression driven by amplicon #161; Merge with DAPI in grey). ( E ) Representative coronal brain section from P7 mouse transduced as in D at P0, but with a reporter vector carrying amplicon #264, a negative control with no predicted enhancer activity that did not display activity in the in vivo STARR-seq screen. Close up of boxed region is shown in the panels on the right. ( F ) Functional validation of enhancer activity in different cell types. Enhancers mDlx and amplicon #161 were individually packaged into reporter constructs and transduced into neonatal mouse brains with a CAG-driven control virus. Brains were collected at P7 and stained for GFP and Ctip2, a transcription factor necessary for axon development in excitatory projection neurons in Layer V during embryonic development. Inset panels show single channel images (Green, EGFP; Red, mRuby3; Magenta, Ctip2). Ctip2 channel images are shown with EGFP + cells outlined (top) and mRuby3+ cells outlined (bottom). Cells that expressed EGFP under the control of the inhibitory interneuron enhancer mDlx displayed a lower frequency of Ctip2 + nuclei compared to cells that drove EGFP under the control of amplicon #161 or drove mRuby3 under the control of the general mammalian promoter CAG.

Article Snippet: We next linearized the vector pscAAV-Hsp68-EGFP ( ) using PacI (NEB #R0547L) and AscI (NEB #R0558L).

Techniques: Functional Assay, Plasmid Preparation, Injection, Expressing, Immunohistochemistry, Fluorescence, Transduction, In Vivo, Amplification, Variant Assay, Activity Assay, Mouse Assay, Staining, Negative Control, Construct

Journal: bioRxiv

Article Title: Parallel functional testing identifies enhancers active in early postnatal mouse brain

doi: 10.1101/2021.01.15.426772

Figure Lengend Snippet: Validation of cell-type specific enhancer function in the STARR-seq orientation. (A, B) Representative confocal images of coronal sections of P7 mouse brain transduced by intracranial injection at P0 with scAAV9-Hsp68-EGFP-mDlx (A) or AAV9-mDlx-βGlobinMinP-EGFP (B) and a CAG-mRuby3 positive control. Sections were stained with an antibody for EGFP for signal amplification. Green, EGFP; red, mRuby3; grey, DAPI. (C) Quantification of the numbers of Pyramidal (light blue), Non-pyramidal (darker blue), and cells of ambiguous morphology (grey) observed in confocal imaging experiment in A and B above; N = 5 animals, 314 cells transduced with Hsp68-EGFP-mDlx (Hsp68-3’UTR) and 4 animals, 972 cells transduced with mDlx-βGlobinMinP-EGFP (βGlobin-5’UTR). (D) Quantification of images from experiment shown in Figure 4F , with mDlx-βGlobinMinP-EGFP included for comparison. Individual GFP+ and mRuby3+ cells were counted and scored for whether each cell contained a Ctip2-positive nucleus. Cell counts were summed across images for the same brain. Data is presented as mean proportion of Ctip2-positive cells (n = 5 animals co-injected with Hsp68-EGFP-#161 and CAG-mRuby3, 4 animals co-injected with Hsp68-EGFP-mDlx and CAG-mRuby3, and 2 animals co-injected with mDlx-βGlobinMinP-EGFP and CAG-mRuby3).

Article Snippet: We next linearized the vector pscAAV-Hsp68-EGFP ( ) using PacI (NEB #R0547L) and AscI (NEB #R0558L).

Techniques: Injection, Positive Control, Staining, Amplification, Imaging, Transduction

Journal: bioRxiv

Article Title: Parallel functional testing identifies enhancers active in early postnatal mouse brain

doi: 10.1101/2021.01.15.426772

Figure Lengend Snippet: Functional dissection of the large third intron of CACNA1C . ( A ) UCSC Genome Browser representation of amplicons #1 through #7 in the third intron of CACNA1C (hg38, chr12:2,220,500-2,242,499). Normalized coverage of aligned reads for DNA and RNA samples are shown for the four biological replicates. Normalized coverage of aligned reads for DNA is also shown for the pre-viral plasmid library. Three amplicons, #3, #6, and #7, were found significantly active in our assay. ( B ) Confocal images of single-candidate validation of amplicons #2, #3, and #6 (red and blue highlighted amplicons in A). Mice were transduced at P0 with two AAV vectors: one for an HSP68-EGFP-3’UTR enhancer reporter construct carrying the indicated amplicon and a second control vector, CAG-mRuby3. Brains were fixed at P7 and sectioned and stained with an antibody for EGFP for signal amplification. Tiled, whole section images are shown on the left. Close up of boxed regions are shown in the panels on the right. Green, EGFP; red mRuby3; grey, DAPI. These experiments validated robust EGFP expression driven by the two positive MPRA hits (#3 and #6), with substantial EGFP reduction for the MPRA negative amplicon #2. ( C ) Mice were transduced with AAV including positive amplicon #3 and processed as in B, but were raised to P28 before fixing, sectioning, and staining.

Article Snippet: We next linearized the vector pscAAV-Hsp68-EGFP ( ) using PacI (NEB #R0547L) and AscI (NEB #R0558L).

Techniques: Functional Assay, Dissection, Plasmid Preparation, Mouse Assay, Construct, Amplification, Staining, Expressing, Transduction