Module 2: Molecular Biology and Virus Production
The Molecular Biology and Virus Production (MB/VP) Module generates and validates DNA constructs primarily for use in mammalian cultures/systems in vision research. This module also purifies and concentrates both lentivirus and adenovirus for subsequent use in immortalized and primary cultures (maintained by individual investigators and the Cell Culture & Cell Phenotyping Module). The MB/VP Module provides the following services:
1) De novo generation of constructs to be used for vision research
2) Modification (mutagenesis, insertion/deletion) of plasmid DNA
3) Maintenance and propagation of DNA glycerol stocks
4) Production and purification of lentivirus and adenovirus
5) Validation of established DNA constructs and lentivirus/adenovirus by immunocytochemistry, qPCR and western blotting
The specific services provided within the MB/VP Module are:
- Site-directed mutagenesis for generating point mutations, insertions, or deletion variants
- Custom Gibson assembly for development of new DNA fusion constructs driven by desired promoters of interest
- ShRNA and CRISPR/SpCas9 plasmid generation for knockdown/out experiments
- Full sequencing of generated constructs, extensive documentation, and maintenance of DNA stocks for future use
- Small to large scale endotoxin free plasmid purification for downstream applications
- Thorough confirmation of produced constructs at the transcript, protein and subcellular level
- Generation of high-purity lentivirus and adenovirus for use in vitro or in vivo
Overall, the MB/VP Module supports a range of enabling recombinant DNA technologies and provides the assistance and expertise required to test unique hypotheses posited by vision scientists at UTSW and neighboring institutions.
The Molecular Biology and Virus Production (MB/VP) Module laboratories are on the 7th floor of the Florence (E) Building in close proximity to the laboratories in the Department of Ophthalmology. The Facility is housed in five rooms (all within 2 minute walk of each other, each dedicated to the specific Core function) having a total of 1610 ft2 of space. The MB/VP Module contains resources for DNA synthesis, mutagenesis, purification, sterile cell culture, ultracentrifugation, and transcriptional/proteomic evaluation. Desk space for the Technical Support staff is located in rooms adjacent to the Facility (E7.239, E7.242).
- Molecular Biology Laboratories (E7.132 and E7.239; 500 ft2)
These two labs have the following equipment that is relevant to the day-to-day function of this module: a clean bench, four PCR machines (2 of which are capable of gradient PCRs), 4 independent gel rigs and power supplies, a LI-COR Fc for imaging DNA gels, a Safe Imager for excising DNA bands, two shaker incubators, three dry baths of varying temperature, one water bath, one refrigerated thermal mixer, one refrigerator, one -20° freezer.
- Viral Production Laboratories (E7.130, E7.238; 960 ft2)
These two labs have the following equipment that is relevant to the day-to-day function of this module: two BSL2 biosafety cabinets, four cell culture incubators, one Heracell cell culture shaking incubator, a Sorvall floor model centrifuge, a Sorvall ultracentrifuge, a refrigerated minicentrifuge, two tabletop centrifuges (one refrigerated), one refrigerator, one -80 freezer. Biohazard-related safety: All personnel handling biohazardous material will receive proper training from UT Southwestern Office of Safety and Business Continuity. All virus work is performed using BSL3 safety practices including containment of potential aerosol formation during centrifugation/agitation, and decontamination of cultures and surfaces using 20% bleach and/or 1% Virkon prior to discarding. Due to the scale of lentivirus/adenovirus produced, the MB/VP Module periodically tests for replication competence through commercially available RCA assays (Cell BioLabs, MyBioSource).
- Additional Supporting Instrumentation: (E7.239, 150 ft2 in E7.212)
Nanodrop for DNA/RNA/protein quantification [located in the common equipment lab, E7.212]. LI-COR CLx for imaging western blotting after transient transfection [located in the lab of Dr. Hulleman, E7.239]. QuantStudio6 to confirm expression levels by qPCR after transient transfection [located in the common equipment lab, E7.212]. Additional -80° freezer space [E7.212] for backup storage of single use viral aliquots.
- Additional Access to UTSW Core Facilities
In addition to the services provided directly by the MB/VP Core (described above), this Core utilizes, or facilitates the utilization of, established UTSW Cores. All sequencing is performed by the UTSW McDermott Sanger Sequencing Core which enables next business day results (drop off box located in adjacent L building, picked up multiple times daily), and appropriate AAV vectors (along with required replication/capsid plasmids) are generated and large-scale purified for eventual packaging by the UTSW AAV core, north campus, NA2.500 (UTSW Viral Vector Facility, directed by Dr. Steven Gray).
Currently, when eye researchers at UTSW require recombinant DNA synthesis or lentivirus/adenovirus production, they must either outsource their efforts to outside companies or identify laboratories that are willing and able to accommodate their requests. Neither option is ideal, and can cause significant delays in projects as well as financial burdens. No Core facility currently exists at UTSW to produce recombinant DNAs or lentivirus/adenovirus. The Hulleman Laboratory generates new DNA constructs on a weekly basis and routinely generates lentivirus/adenovirus for use in cell culture studies. The Hulleman Lab has collaborated (as evidenced by publications) with the Mootha Lab at UTSW and the Zode Lab at UNTHSC to perform the exact services described in this Module.
The UTSW Department of Ophthalmology has demonstrated a commitment to supporting research described in this module through the recent purchase of instrumentation such as the LI-COR CLx, QuantStudio6, and a Sorvall ultracentrifuge. These pieces of equipment, as well as others described below are currently under Dr. Hulleman’s supervision, and have the bandwidth to accommodate the proposed studies without affecting productivity of current Ophthalmology Department users, including the Hulleman Lab. Additional dedicated space (in E7.130) for cell culturing related to the proposed services is available to Dr. Hulleman for this Module.
- Personnel and Administration
The Molecular Biology/Virus Production (MB/VP) Module is led by Dr. John Hulleman who has over 16 years of relevant experience in molecular biology, protein analysis and virus production techniques. The day-to-day operation of the module has been assigned to one full-time technician, Ms. Marian Renwick, and one half-time technician. The full-time technician is responsible for: 1) generating newly requested DNA constructs, 2) maintaining glycerol stocks, performing full sequencing of constructs, establishing individual plasmid maps, and 3) processing appropriate scale plasmid preps for subsequent transient transfections, lentivirus and adenovirus production. The half-time technician is responsible for 1) testing newly generated constructs using immunocytochemistry, qPCR and western blotting, 2) producing and purifying lentivirus and adenovirus.
- Module Operations
The technical support staff performs routine maintenance, ensuring that all equipment is in proper working order and attempts to identify specific service needs before equipment fails. Service contracts are maintained for all major pieces of equipment to ensure limited down time following equipment failure.
Requests for technical support and hands-on assistance from the MB/VP Module staff to conduct experiments will be submitted to Ms. Renwick for review by the Director. These requests include the purpose of the study, number of samples to be generated and the ultimate goals of the project. Prior to initiation of a project, Dr. Hulleman, Ms. Renwick and the individual investigator will meet to discuss the ultimate goals and Dr. Hulleman will establish a research plan with the best route forward for achieving said goals. Once a research plan has been established, it will be transferred to a request form to initiate the work along with a given timeframe for the work. Each individual investigator will receive weekly updates once their project has started. Projects will be prioritized according to initiation date, but consideration will also be given based on the ultimate goal of the project (i.e., if constructs will be used for preliminary studies vs. funded R01 projects).
Problems with the operation of this module are troubleshot by Dr. Hulleman, and these problems, if persistent, are addressed in the monthly Center Director’s meeting. A logbook and sign up record are maintained for each major piece of equipment. All electronic data are stored on a secure network drive that is backed up on a daily basis. As it relates to studies performed under this module, only Ms. Renwick and the technician use the identified equipment. Only under specific requests are additional personnel (from the Investigators’ laboratories) trained on how to perform the described techniques or on how to use the instrumentation.
- Services and Methodologies
Recombinant DNA synthesis has been a routine laboratory task for > 25 years, and as such, it is a critical foundation for exploring new ideas of how to manipulate and/or restore biology. It goes without saying that even single base pair alterations (synonymous or mutations) can alter the encoded protein. Thus, it is critical to maintain pristine records to ensure DNA fidelity and rigor/reproducibility of subsequent experiments. Yet, in practice, this can be a painstaking and laborious task requiring dedicated personnel to ensure consistency and maintain standards. The MB/VP Module enables users to generate virtually any construct for their studies. These constructs can then be used for either overexpression or knockdown/out studies. DNA constructs are fully sequenced and stocks (glycerol and plasmid) are well documented and maintained in Microsoft Access spreadsheets and SnapGene files. Additional contributions that this module makes to ensure rigor and reproducibility include validation of the produced DNA constructs at the transcript level using highly specific TaqMan probes and at the protein level (western blot and immunocytochemistry) using knockout/down validated antibodies. Furthermore, centralizing and confining lentivirus and adenovirus production into a single facility minimizes expenses of maintaining sterile BSL2 facilities and maximizes containment of infectious biohazard material.
- Generation of New DNA Constructs
For projects to test expression of a gene of interest in any given system, we first check the Hulleman Lab DNA repository which contains ~850 unique DNA constructs or open sources (like Addgene, DNASU) that provide access to >250,000 unique plasmids in a variety of species. If cDNA cannot be obtained from the above sources, we generate the cDNA using PCR amplification from target tissue mRNA. The cDNA of interest is cloned (ligated) into the pENTR1A Gateway Entry Vector (Life Technologies) using appropriate restriction sites. All DNA clones are verified fully by sequencing (including the promoter and 3’ UTR, if applicable, McDermott Sanger Sequencing Core). Using the pENTR1A clone, we introduce appropriate tags (such as HA, FLAG, myc) using Q5 mutagenesis (NEB) and appropriate NEBulider-designed primers (NEB). For insertions up to 30 nucleotides, or any size deletion, the Q5 mutagenesis kit is also used. For larger insertions and multiple gene assemblies into one construct, Gibson Assembly (NEB HiFi Master Mix) is used. If a construct cannot be appropriately synthesized by these means, we purchase a gBlock oligonucleotide of the sequence, ligate it into pENTR1A and screen the resulting clones to identify the correct sequence. Each correct pENTR1A clone is stored as a glycerol stock for safekeeping and a SnapGene file is generated for each investigator (each investigator is also given a software license of the full version of SnapGene to allow for viewing and manipulation of the sequence).
The pENTR1A constructs are shuttled into one of 5 different vectors depending on ultimate need; pcDNA-DEST40 (constitutive CMV mammalian expression), pTRex-DEST30 (doxycycline-regulated CMV-driven mammalian expression), pLenti4-TO-DEST (doxycycline-regulated CMV-driven mammalian expression using lentivirus), pLenti6-V5-DEST (CMV-driven mammalian expression using lentivirus), or pAD-V5-DEST (constitutive CMV mammalian expression using adenovirus) by LR Clonase II reaction. An alternative pCAG (chicken b actin/CMV hybrid promoter) vector is used as a destination vector if we find silencing of the CMV promoter in the above vectors based on subsequent follow up experiments in mammalian cells. Additionally, if desired, the constructs can be ligated into adeno-associated virus (AAV) vectors for ultimate production by the UTSW Viral Vector Facility, which can produce laboratory-grade AAV at minimal cost.
For long term knockdown, shRNA is a preferred method to specifically reduce transcript levels. We purchase and test a series of validated, commercially-available shRNA clones against transcripts of interest (Sigma) in lentivirus vector backbones driven by either the U6 or H1 promoter. These constructs are validated in cell culture for knockdown efficiency (qPCR and protein level) and for potential off target effects (by examining levels of highly homologous genes, if present). For knockout studies, we design a series of guide RNAs (using Benchling software), insert them into a conventional all-in-one vector (such as PX459 v2.0, Addgene) and test their ability to generate in/dels using the SpCas9 nuclease by a T7E1 assay (NEB). Promising gRNA leads are then prioritized for future in vitro or in vivo experiments.
- Differential Scale DNA Plasmid Preparation
After fully validating the destination constructs by sequencing and restriction digestion (typically mini preparations are sufficient), we then scale up the culture volume to achieve midi preparation scale (for typical transient transfection experiments and adenovirus production), or maxi preparations (for lentivirus production). Qiagen Midi/Maxi Prep Plus kits (which reduce the amount of endotoxin in the resulting DNA) are used. Confirmation of low endotoxin levels is confirmed using the Tissue Culture and Cellular Phenotyping module. Final DNA concentration is determined by Nanodrop and verified on a gel, again using restriction digestion.
- Verification of DNA Construct Expression and Correct Subcellular Localization
Prior to giving the new DNA construct to the individual investigator, we verify that it expresses appropriately in basic 293A-based cell cultures. 293A cells are plated overnight in 6 well dishes to yield enough cellular material for western blotting or qPCR. Cells are transfected using Lipofectamine 3000. Cells used for western blotting are lysed in RIPA buffer supplemented with benzonase and protease inhibitor, normalized by BCA and denatured on an SDS-PAGE followed by transfer to a nitrocellulose membrane. Western blotting imaging is performed on a LI-COR CLx using conventional, validated antibodies to the protein of interest, or an appropriate epitope tag. Cells for qPCR analysis are processed using an Aurum Total RNA kit. Samples are reverse transcribed and evaluated for mRNA transcript using TaqMan probes (Life Technologies). Immunolocalization studies are performed by transfecting 293A cells on glass bottom-plates, followed by fixation, antibody incubation and imaging on a confocal microscope using the Microscopy and Digital Imaging Module. Fluorescent organelle markers (purchased as DNA constructs from Addgene) are used as positive controls. After validation of these three parameters, constructs, along with all sequencing data and SnapGene files are provided to the individual investigators for their studies. If mislocalization or poor expression is observed, we attempt to transfect the construct in a more physiologically relevant cell type (like ARPE-19, 661W, etc.) in case additional protein partners are necessary for production.
- Production/Purification of Lentivirus and Adenovirus
After verification of protein expression, mRNA production and correct subcellular localization, if necessary, replication incompetent lentivirus are produced by triple transfection of 5 x 10 cm dishes of 293T cells with the plasmid of interest, VSVG and PSP plasmids using Lipofectamine 3000. Media are harvested at 48 and 72 h and pooled. Lentivirus is then purified by sucrose gradient ultracentrifugation and viral particles collected. Viral titers are calculated by p24 ELISA (Abcam) as well as functional titers using standard plaque assays/crystal violet staining. Lentivirus is aliquoted and frozen at -80° in single use aliquots to avoid freeze thaw-mediated reduction in titers.
Alternatively, adenovirus is generated by linearization of the adenovirus DNA with PacI (absence of PacI sites in the gene of interest is validated prior to ultimate generation of adenovirus) followed by transfection into 293A cells in a 6 well plate. Three days after transfection, cells are replated into 10 cm dishes, followed by media changes until cytopathic effects (CPE) are observed. Once near 100% CPE is observed, cells and media are frozen/thawed to release the adenovirus. One-hundred mL of P1 virus is used to amplify viral load and make P2 virus in a similar fashion. P3 and P4 virus is made, and high-titer P4 virus is collected, released by cells via freeze/thaw and purified using column affinity (Virapur), concentrated and eluted. Adenovirus is tittered by an Adenovirus Rapid Quantitation Kit (Cell Biolabs) and tittered functionally by a plaque assay. Adenovirus is aliquoted and stored at -80° until use.