Sanford Burnham Prebys Medical Discovery Institute

AAV, lenti-, retro-, adeno-, vaccinia-, zika-, corona-, sindbis virus, and VSV

1. Custom packaged lenti-viral (HIV-1 and FIV) production is based on constructs provide by the customer. Titration is offered for virus ubiquitously expressing fluorescent marker.

2. Ready-to-transduce lentiviral preps - include: (1) constitutively expressed or inducible fluorescent protein, antibiotic selection marker, or bioluminescence reporter (2) nuclear or membrane localization tool (3) constitutively expressed non-silencing shRNA control.
   • hPGK-eGFP
   • hPGK-mCherry
   • hPGK-H2B-eGFP (nuclear eGFP)
   • hPGK-H2B-mCherry (nuclear mCherry)
   • hPGK-tdTomato
   • hPGK-Firefly Luciferase
   • hEF1α- Firefly Luciferase
   • UBC-eGFP-Firefly Luciferase fusion
   • CMV-Renilla-Luciferase
   • Tet-inducible-eGFP
   • Tet-inducible-tdTomato
   • CMV-NLS-eGFP (nuclear eGFP)
   • CMV-NLS-mCherry (nuclear mCherry)
   • CMV-eGFP-CAAX (membrane eGFP)
   • CMV-mCherry-CAAX (membrane mCherry)
   • LKO.1-scrambled shRNA (Sigma-Aldrich)
   • GIPZ-scrambled shRNA (Open Biosystems)
   • TRIPZ-scrambled shRNA (Open Biosystems)

3. "The Works" custom lentiviral vector construction – offers vector generation based on cDNA or shRNA of interests with a choice of constitutive/inducible expression vectors, viral production, and titration.

4. Custom viral particles concentration and purification with user-provided viral supernatants allows customers to increase viral titer (up to 1000 fold) based on crude viral supernatants.

1. Custom packaged retro-viral (MMLV) production is based on constructs provide by the customer. Titration is offered for virus ubiquitously expressing fluorescent marker.

2. Ready-to-transduce retroviral preps - include: constitutively expressed fluorescent protein.
   • GFP
   • mCherry

1. Custom production of adenoviral particles (Ad5) from user-supplied construct or virus - includes: (1) adenovirus recovery (2) high purity adenoviral preparation using CsCl gradient purification (3) titration using the plaque assay.

2. "The Works" custom lentiviral-adenoviral vector construction and swapping - includes: (1) constitutive expression of custom cDNAs (2) inducible shRNA of interests expressed under a new Tet inducible system (3) Custom promoters/enhancer reporter vectors (4) MicroRNA sponges.

1. "The Works" custom AAV vector construction and virus packaging
   • Serotype Tropism
    AAV-1 : SM, CNS, retina, pancreas
    AAV-2 : In vitro, VSMC, SM, CNS, liver, kidney
    AAV-3 : Hepatocarcinoma, SM
    AAV-4 : CNS, retina, lung
    AAV-5 : SM, CNS, lung, retina
    AAV-6 : SM, SM (i.v.), heart, lung
    AAV-8 : Liver, SM, CNS, retina, pancreas, heart
    AAV-DJ : In vitro, liver, heart, kidney
    AAV-DJ/8 : liver, heart, kidney, pancreas, SM, brain (i.v.)

   • Ready-to-transduce AAV
    AAV-2-GFP
    AAV-DJ-GFP

1. "The Works" custom VSV vector construction and production
    Pseudotyped VSV-ΔG-GFP
    Ebola virus glycoprotein (EboV-GP) Zaire strain (Kissidougou-C15, 2014)
    Vesicular stomatitis virus glycoprotein (VSV-G)
   Example: VSV/Lenti-GFP-EboV-GP expression

1. Custom packaged Sindbis viral vector production is based on constructs provide by the customer.

1. All-in-one CRISPR/Cas gene disruption and editing viral vectors
   • Knock-out using viral vector platform (hCas9 + gRNA)
    Gene disruption using adenovirus or lentivirus (on demand)
   Example: T7EI assay, gRNA-T1 to 3 targeting Rosa26 loci
   Example: Ready-to-transduce Ad5-CRISPR all-in-1 targeting GFP

   • Knock-in using plasmid-free viral vector platform (hCas9 + gRNA + Donor DNA)
    Gene editing using adenovirus (on demand)
    Human AAVS1 safe harbor loci
    Mouse Rosa26 safe harbor loci
   Example: knock-in GFP-2A-Gene of interests in Rosa26 loci

2. Genetic modified mouse creation
   • Knock-in (hCas9 + gRNA + Donor DNA)
    Rosa26 loci
    Choice of C57BL/6 C2 and 129 mESC.
   Example: knock-in GFP-2A-Gene of interests in Rosa26 loci
   gRNA#1: 4 out of 4 screened mESC clonal line(100%) show Rosa loci integration
   gRNA#2: 3 out of 4 screened mESC clonal line (75%) show Rosa loci integration
   gRNA#3: 2 out of 4 screened mESC clonal line (50%) show Rosa loci integration

3. Custom CRISPR gene inhibition (CRISPRi) and activation (CRISPRa)
   • CRISPRi viral vector delivery
    Adenovirus (dCas9-Krab)
    Lentivirus (dCas9-Krab and gRNA)
   Example: SOX2 CRISPRi

   • CRISPRa viral vector delivery
    Lentivirus (dCas9-VP64 and gRNA)

1. Custom lenti- and adeno-viral reporter for 2D and 3D screening
   • One secreted luciferase is constitutively expressed by hPGK promoter for normalization.
   • Another secreted luciferase is expressed by your promoter of interest.
   • The third secreted marker is expressed by another promoter of interest, your choice.
   Example: GFAP expression*

1. Large scale DNA plasmid preparation (ready for viral production) starts from a small amount of customer's vector (e.g. miniprep) or transformed bacterial stock.