Useful Information

The facility is committed to educating its users. As such, the following links provide information that users may find helpful about the services provided by the facility.

Molecular Screening Links
Protein Expression Links

Molecular Screening

shRNA Ordering Guidelines

NCGC assay Guidance manual
http://ncgc.nih.gov/guidance/manual_toc.htm

General Assay development
High-throughput screening assays for the identification of chemical probes. Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. Nat Chem Biol. 2007 Aug;3(8):466-79. PMID: 17637779

Luciferase based screens
Characterization of chemical libraries for luciferase inhibitory activity.Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, Austin CP, Inglese J. J Med Chem. 2008 Apr 24;51(8):2372-86. Epub 2008 Mar 26. PMID: 18363348

Fluoresence: Intensity, anisotropy/polarization (FA/FP)
Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. Pope AJ, Haupts UM, Moore KJ. Drug Discov Today. 1999 Aug;4(8):350-362. PMID: 10431145

Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. Owicki JC. J Biomol Screen. 2000 Oct;5(5):297-306. PMID: 11080688

Real experiences of uHTS: a prototypic 1536-well fluorescence anisotropy-based uHTS screen and application of well-level quality control procedures. Turconi S, Shea K, Ashman S, Fantom K, Earnshaw DL, Bingham RP, Haupts UM, Brown MJ, Pope AJ. J Biomol Screen. 2001 Oct;6(5):275-90. PMID: 11689128

Aggregation based inhibition
A high-throughput screen for aggregation-based inhibition in a large compound library. Feng BY, Simeonov A, Jadhav A, Babaoglu K, Inglese J, Shoichet BK, Austin CP. J Med Chem. 2007 May 17;50(10):2385-90. PMID: 17447748

Thermal stability
Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Lo MC, Aulabaugh A, Jin G, Cowling R, Bard J, Malamas M, Ellestad G. Anal Biochem. 2004 Sep 1;332(1):153-9. PMID: 15301960

Data analysis/reporting
A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Ji-Hu Zhang, Thomas D. Y. Chung and Kevin R. Oldenburg (1999). J. Biomol. Screen 4:67-73.

Statistical practice in high-throughput screening data analysis. Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R. Nat Biotechnol. 2006 Feb;24(2):167-75. PMID: 16465162

Reporting data from high-throughput screening of small-molecule libraries. Inglese J, Shamu CE, Guy RK. Nat Chem Biol. 2007 Aug;3(8):438-41. PMID: 17637769

Chembank
http://chembank.broadinstitute.org

Pubchem
http://pubchem.ncbi.nlm.nih.gov

 

Protein Expression

Baculovirus Expression Systems
Lentivirus Preparation

Vector Database

FAQ

Baculovirus Expression Systems

DNA Transfection for Baculovirus Expression Vector System 

Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector (donor or shuttle) plasmid DNA containing the foreign gene to be expressed and BaculoGold™ DNA (PharMingen), Bac-N-Blue™ DNA (Invitrogen), or BacPAK6™ DNA (Clontech).  Alternatively, insect cells are transfected with a recombinant bacmid DNA constructed by transposition of the donor plasmid DNA in E. coli cells, the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method. The baculovirus DNA is from Autographica californica nuclear polyhedrosis virus (AcNPV).

The BaculoGold™ and Bac-to-Bac™ methods are designed to achieve virtually 100% recombination efficiencies and recombinant protein expression is subsequently evaluated using recombinant virus amplified (without plaque purification) in P2 in insect cells. A single plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) or higher. Recombinant baculovirus derived from all other commercially available baculovirus DNA preparations is produced with 80-90% efficiency and requires plaque purification to remove parental virus. 

Recombinant Baculovirus Stocks

High titer P2 virus stock is produced from the P1 virus (the original virus from cell culture supernatant of co-transfected cells) in Sf9 cells.  P2 virus stock is generally produced in 100 ml of serum-free medium.  A P3 virus (50 ml or 500 ml) is produced by infecting Sf9 cells with a P2 virus at a low multiplicity of infection (MOI=0.1).

Plaque Assay and Purification of Recombinant Baculovirus

Plaque assays are done by infecting sf9 or sf21 insect cells with the P1 virus (or higher passage virus) at a low multiplicity of infection (MOI = 0.1) and overlaying the infected cells with agarose. Well isolated plaques are scored and virus is subsequently amplified in monolayer cultures of Sf9 cells prior to preparation of larger volume high-titer stocks.

Time-Course Study of Protein Expression 

100 ml suspension cultures of insect cells are infected with a high-titer baculovirus stock at an MOI=1-2.  Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection to evaluate the integrity, stability and optimum yield of the product(s) of gene expression. The results can be used to determine optimal conditions for protein production. Harvested samples are analyzed for expression of the recombinant protein by western blot.  The staff can compare recombinant protein production in at least three different cell lines (sf9, sf21, and High Five) if necessary.

Large-Scale Production of Recombinant Proteins

High density (1-2 x106 cells/ml) suspension cultures of Sf9, Sf21, or Trichoplusia ni (commonly referred to as High Five) cells are infected at a multiplicity of infection (MOI) equal to 1-2. High Five cells are preferred for production of secreted proteins. We routinely infect cultures (250 ml to 1 L) of insect cells in serum-free medium containing Pluronic F-68 (to prevent cell damage due to shearing) in spinner bottles. 
For additional information about insect cell culturing and baculovirus expression systems, please see:
          Guide to baculovirus expression systems and insect cell culturing (PDF)
          Bac-to-Bac Baculovirus expression systems (PDF)
          Baculodirect Baculovirus Expression Systems (PDF)
          Bac-N-Blue Baculovirus Expression System (PDF)

Lentivirus Preparation

HIV-derived lentiviral vectors have been developed as a gene delivery system that can mediate the efficient delivery, integration, and sustained long-term expression of transgenes to dividing and non-dividing cells in vitro and in vivo. Over the years, lentiviral vectors have turned out to represent very powerful tools in basic and translational scientific research. As such, the facility has implemented the infrastructure and technology to produce high-titer lentiviral vectors. In addition, we have pursued their technological improvement for constitutive and conditional expression of cDNAs and shRNAs.

Lentivirus particles are generated by co-expressing the virion packaging elements and the vector genome 
via transient transfection in a producer cell line (e.g. HEK293T). For HIV-1-based vectors, the core and enzymatic components of the virion come from HIV-1, 
while the envelope is derived from a heterologous virus, most often vesicular stomatitis virus (VSV) 
due to the high stability and broad tropism of its G protein. 
By convention, the former elements are referred to as the LV packaging system and the latter as the envelope.

3 components are required to produce an infectious lentivirus vector:
• vector (e.g., pLU, pLKO, pGIPZ)
• packaging system (e.g., pCMV-dR8.91, psPAX2 (2nd generation))
• envelope plasmid, (e.g., pMD2G (VSVg))

Three generations of HIV-based LV packaging systems have been developed for production of lentivectors by transient transfection.

The first generation LV packaging system encompasses all HIV-1 genes besides the envelope.

The second generation LV packaging system is additionally deleted in all viral auxilliary genes, 
i.e., vpr, vif, vpu and nef. Examples: pCMV-dR8.91, pCMV-dR8.74, psPAX2

The third generation LV packaging system comprises only gag, coding for the virion main structural proteins and pol, responsible for the retrovirus-specific enzymes. A cDNA encoding rev, which encodes a post-transcriptional regulator necessary for efficient gag and pol expression, is provided on a separate plasmid. The third generation packaging system offers maximal biosafety but is more cumbersome, involving the transfection of four different plasmids in the producer cells.
Plasmid DNAs for the 3rd generation packaging system include pMDL g/p RRE and pRSV-Rev.

All lentiviral vectors that contain a wild-type 5'LTR (e.g., pGIPZ) need to be packaged with a 2nd generation packaging system, as wt 5'LTR requires TAT for maximal activation of transcription. The 3rd generation packaging system can only be used with a lentiviral vector with a chimeric 5'LTR e.g. CCL-, RRL-, etc, in which HIV promoter has been replaced with CMV or RSV, thus making them TAT-independent. The lentivectors carrying the chimeric 5'LTR can be packaged into both, 2nd or 3rd generation packaging system.

The production of vector particles by our facility uses a second generation packaging system, which satisfies most applications.

The vector itself is the only genetic material transferred to the target cells. It typically comprises the transgene cassette flanked by cis-acting elements necessary for its encapsidation, reverse transcription and integration. As previously done with oncoretroviral vectors, advantage was taken of the gymnastics of reverse transcription to engineer self-inactivating (SIN) HIV-1-derived vectors, which lose the transcriptional capacity of the viral long terminal repeat (LTR) once transferred to target cells. This minimizes the risk of emergence of replication 
competent recombinants (RCR) and avoids problems linked to promoter interference.

Vector Database

Bacterial Expression Plasmids

plasmid name

vendor

Tags

Ab

 

 

 

pRSETa

Invitrogen

N-6His

Amp

map

mcs

seq

pRSETb

Invitrogen

N-6His

Amp

map

mcs

seq

pRSETc

Invitrogen

N-6His

Amp

map

mcs

seq

pGEX-2T

GE Healthcare

N-GST

Amp

map

mcs

seq

pGEX-4T-1

GE Healthcare

N-GST

Amp

map

mcs

seq

pGEX-4T-2

GE Healthcare

N-GST

Amp

map

mcs

seq

pGEX-5X-1

GE Healthcare

 

Amp

map

mcs

seq

pGEX-5X-3

GE Healthcare

N-GST

Amp

map

mcs

seq

pGEX-6P1

GE Healthcare

N-GST

Amp

map

mcs

seq

pQE30

Qiagen

N-6His

Amp+Kan

map

mcs

seq

pQE31

Qiagen

N-6His

Amp+Kan

map

mcs

seq

pQE32

Qiagen

N-6His

Amp+Kan

map

mcs

seq

pQE50

Qiagen

 

Amp+Kan

map

mcs

seq

pET28a

Novagen

 

Kan

map

mcs

seq

pDUET

Novagen

 

 

map

mcs

seq

pMAL2CE

NEB

MBP

Amp

map

mcs

seq

Baculovirus Expression Vectors

Bac-to Bac method

 

 

 

 

 

 

 

plasmid name

vendor

tag

cleavage secretion  

pFastBAC 1

Invitrogen

 

 

map

mcs

seq

pFastBAC Dual

Invitrogen

 

 

map

mcs

seq

pFastBAC HTA

Invitrogen

N-6His

Tev

map

mcs

seq

pFastBAC HTB

Invitrogen

N-6His

Tev

map

mcs

seq

pFastBAC HTC

Invitrogen

N-6His

Tev

map

mcs

seq

pFastBAC GST

Invitrogen

N-GST

Tev

map

mcs

seq

pFastBAC Flag

Invitrogen

N-FLAG

Tev

map

mcs

seq

pFastBAC Strep/His

Invitrogen

N-StrepII/6His

Tev

map

mcs

seq

pFastBAC FLAG/HTA

Invitrogen

N-FLAG/6His

Tev

map

mcs

seq

pFastBAC-HTB/FLAG

Invitrogen

N-6His/FLAG

Tev

map

mcs

seq

pFastBAC C-FLAG/His

Invitrogen

C-FLAG/His

Tev

map

mcs

seq

 

Baculogold method

 

 

 

 

 

 

plasmid name

vendor

tag

cleavage secretion

  

pVL 1392

Pharmingen

 

 

 

map

seq

pVL 1393

Pharmingen

 

 

 

map

seq

pAC HLT a

Pharmingen

N-6His

thrombin

 

map

seq

pAC HLT b

Pharmingen

N-6His

thrombin

 

map

seq

pAC HLT c

Pharmingen

N-6His

thrombin

 

map

seq

pAC GHLT a

Pharmingen

N-GST/6His

thrombin

 

map

seq

pAC GHLT b

Pharmingen

N-GST/6His

thrombin

 

map

seq

pAC GHLT c

Pharmingen

N-GST/6His

thrombin

 

map

seq

pAC GP67 a

Pharmingen

 

 

X

map

seq

pAC GP67 b

Pharmingen

 

 

X

map

seq

pAC GP67 c

Pharmingen

 

 

X

map

seq

pAc secG2T

Pharmingen

N-GST

thrombin

X

map

seq

pAc GP67b-His

Pharmingen

N-6His

Tev

X

 

 

 

 

 

 

 

 

 

pPolh FLAG

Sigma

FLAG

 

 

map

seq

 

 

 

 

 

 

 

Bac-N-Blue method

 

 

 

 

 

 

pBlueBAC 4.5 V5/HIS

Invitrogen

C-V5/6His

 

 

map

seq

Mammalian Expression Vectors

plasmid name

vendor

promoter

tag

selection

 

 

pcDNA3

Invitrogen

CMV

 

Neo

map

seq

pcDNA3-FLAG

Invitrogen

CMV

N-FLAG

Neo

map

seq

pcDNA3-HA

Invitrogen

CMV

N-HA

Neo

map

seq

pcDNA3-nGFP

Invitrogen

CMV

N-GFP

Neo

map

seq

pcDNA3-cGFP

Invitrogen

CMV

C-GFP

Neo

map

seq

pcDNA3-dsRED2

Invitrogen

CMV

N-dsRED2

Neo

map

seq

pcDNA3-nYFP

Invitrogen

CMV

N-YFP

Neo

map

seq

pcDNA3-nCFP

Invitrogen

CMV

N-CFP

Neo

map

seq

pcDNA3-GAL4(DBD)

Invitrogen

CMV

N-GAL4

Neo

map

seq

pcDNA3-ERHBDTM

Invitrogen

CMV

N-ERHBD

Neo

map

seq

pcDNA3.1-myc/his (A/B/C)

Invitrogen

CMV

C-myc/his

Neo

map

seq

pcDNA3.1-hygro

Invitrogen

CMV

 

hygro

map

seq

 

 

 

 

 

 

 

pcDNA4(TO)-myc/his (A/B/C)

Invitrogen

CMV-TRE

C-myc/his

Zeocin

map

seq

pcDNA4(TO)-FLAG

Invitrogen

CMV-TRE

N-FLAG

Zeocin

map

seq

pcDNA4(TO)-HA

Invitrogen

CMV-TRE

N-HA

Zeocin

map

seq

pcDNA4(TO)-EGFP

Invitrogen

CMV-TRE

N-EGFP

Zeocin

map

seq

pcDNA4(TO)-V5/FLAG

Invitrogen

CMV-TRE

C-V5/FLAG

Zeocin

map

seq

pcDNA4(TO)-cEGFP

Invitrogen

CMV-TRE

 

 

map

seq

pcDNA6/TR

Invitrogen

 

 

Blasticidin

map

seq

Lentivirus vectors

plasmid name

promoter

tag

selection

 

 

pLU-GFP

CMV

 

GFP

map

seq

pLU-DsRed2

CMV

 

DsRed2

map

seq

pLU-mCherry

CMV

 

mCherry

map

seq

pLU-YFP

CMV

 

YFP

map

seq

pLU-CFP

CMV

 

CFP

map

seq

 

 

 

 

 

 

pLU-TREmin-GFP

TREmin

 

GFP

map

seq

 

 

 

 

 

 

pLU-EF1-GFP

EF1

 

GFP

map

seq

pLU-EF1-mCherry

EF1

 

mCherry

map

seq

pLU-EF1-FFluc

EF1

 

 

map

seq

pLU-EF1-Ffluc/mCherry

EF1

 

mCherry

map

seq

 

 

 

 

 

 

pLU-UbiC-GFP

UbiC

 

GFP

map

seq

pLU-UbiC-mCherry

UbiC

 

mCherry

map

seq

 

 

 

 

 

 

pLU-T-CMV-pPuro

TRE-CMV

N-FLAG

Puro

map

seq

pLU-T-CMV-pBlast

TRE-CMV

N-FLAG

Blasticidin

map

seq

pLU-T-CMV-pGFP

TRE-CMV

N-FLAG

GFP

map

seq

 

 

 

 

 

 

pLU-TREmin-pPuro

TREmin

N-FLAG

Puro

map

seq

 

 

 

 

 

 

pLU-T-EF1-pPuro

TRE-EF1

N-FLAG

Puro

map

seq

pLU-T-EF1-pBLAST

TRE-EF2

N-FLAG

Blasticidin

map

seq

pLU-T-EF1-pGFP

TRE-EF3

N-FLAG

GFP

map

seq

 

 

 

 

 

 

pLU-tTR-KRAB-iRed2

CMV

 

DsRED2

map

seq

pLU-tTR-KRAB-iBlast

CMV

 

Blasticidin

map

seq

pLU-tTR-KRAB-iCherry

CMV

 

mCherry

map

seq

pLU-rtTA3-iCherry

CMV

 

mCherry

map

seq

 

 

 

 

 

 

pLKO.1

U6

 

puro

map

seq

pLKO-tGFP

CMV

 

turboGFP

map

seq

pGIPZ

CMV

 

Puro

map

seq

FAQ

How long does it take to generate a new baculovirus and test expression?

For the Bac-to-Bac and Baculogold methods, our average time to make the virus, amplify a high-titer stock, and test the kinetics of protein expression is about 3-3.5 weeks.  We will notify you by email when we start your time course expression study or production and when you  will be able to collect your samples from the Facility. 

Is it necessary to plaque purify my virus?

It depends upon the baculovirus transfer/donor vector in which you clone your GOI. Vectors in the BaculoGold™ and Bac-to-Bac™ methods are designed to achieve virtually 100% recombination efficiencies and recombinant protein expression is subsequently evaluated using recombinant virus amplified (without plaque purification) in P2 in insect cells. A single plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) or higher. Recombinant baculovirus derived from all other commercially available baculovirus DNA preparations is produced with 80-90% efficiency and requires plaque purification to remove parental virus.

Does plaque purification of Baculogold or Bac-to-Bac derived viruses enhance the expression of the target protein? 

We have looked at a limited number of cases and found that we really do not see significant differences in the expression level (<2-fold) or integrity of a protein expressed from 5 plaque purified viruses derived from a parental polyclonal virus supernatant.   

What sort of yield can I expect?

Due to the intrinsic biochemical properties of each individual protein, we do not guarantee recombinant protein solubility, purity, or yield.  We will provide details of all expression trials, purification strategies, and provide consultation regarding alternative strategies or approaches to solve your protein expression needs.  

How do you control for expression?

The Facility aims to maintain suspension cultures of logarithmically growing Sf9, Sf21, and High Five (T.ni) cells in serum free medium (SFM) with cell viabilities greater than 85%. Deviations from these parameters provide an indication of cell deterioration. Cultures are maintained for no more than ~35 passages before reestablishing new cultures of low passage cells from cryopreserved stocks of the same lot. Infection of insect cells with baculovirus vectors inhibits cell growth and cell viability decreases post-infection. During productions, we monitor total cell numbers and percentage of viable cells as a function of time post-infection.

Why does my protein not express or why are the yields for my protein poor?

Assuming the GOI is properly cloned into the baculovirus transfer, this question is difficult to definitively answer. There are numerous examples where the integrity, stability and biological activity of recombinant proteins vary with time after infection. Therefore, the recommendation of the Facility is that each new baculovirus for a protein be extensively characterized in analytical scale time course expression experiments before proceeding to large-scale productions. 

On rare occasions, we have found that expression of a recombinant protein in insect cells can exist in inclusion bodies.  We recommend that investigators analyze both soluble and insoluble protein fractions from analytical time course expression studies. For secreted proteins, we recommend that investigators analyze the amount of protein in the supernatant and in cell pellets to assess the extent of secreted protein expressed.  

How stable are the baculovirus stocks?

We generally store our high-titer viral stocks at 4°C for up to 6 months. While the stability of some viruses extend beyond 6 months, it has been our experience that the titers of the virus begin to decline. The Facility recommends that a new high-titer virus stock be prepared from existing stocks before the initiation of a new production. In addition, we cryopreserve 1ml aliquots of these stocks at -80°C for long term storage.

Can I use the Facility's equipment for my own cell culture?

In order to avoid cross-contamination, the Facility’s equipment is not be accessible for general use.

Do you purify proteins?

Yes!  The Facility provides analytical and preparative scale, one- or two-step purification of recombinant proteins expressed in bacteria, baculovirus infected insect cells, or mammalian cell lines.  For more information, please see services

What is the titer of retroviral vectors you generate?

In our experience the specific titer of a retroviral vector we produce depends upon the total size of the vector, nature of the insert, and quality of the DNA provided. In general, the smaller the total size of the viral vector the better the titers we have been able to generate. We recommend, whenever possible, that newly produced vectors be titrated. We also offer services to concentrate vector preparations.  Based on our protocols, we routinely achieve titers for conditioned cell supernatant in the range 1 x 105-1 x 106 TU/ml.  For concentrated vector, titers range from 1 x108-1x10TU/ml.

Do I need to register my intended use of retroviruses with my Institutional Biosafety Committee?

The process of virus packing by the Protein Expression Facility is registered with the Wistar Institute Biosafety Committee (approval # 21102382, exp 3/2014).  It is the responsibility of the investigator to register the intended specific use of recombinant DNA technology (i.e. retrovirus use) with Wistar’s Institutional Biosafety Committee (IBC) as required by the most current NIH guidelines.

What percentage of the HIV sequence is retained in the lentivirus vector produced?

Based on DNA sequence comparisons between common LV vectors (e.g. pLKO.1, pLU, pGIPZ) and NCBI accession # K03455, we estimate approximately 10-15%.

How much virus do I need to infect my cells?

The transduction efficiency of tissue culture cell lines is variable. We recommend that investigators determine the transduction efficiency of their specific cells with a titrated virus that expresses a fluorescent protein tracer.

How stable are the retrovirus stocks you prepare?

The stability of each virus should be assessed individually. Based on our experience, conditioned cell supernatants can be stable upto 2 months at 4°C with minimal loss of infectivity.  
  
Does the facility provide service for non-Wistar investigators?

Yes!

How do you prioritize work requests?

The Facility’s staff strives to accommodate all requests in a timely order from receipt of a service request. The facility has the capacity to handle between 50-60 insect cell cultures (any volume) per week. All work requests are initiated on a first-come-basis, with Wistar Cancer Center members receiving priority, and all other user requests being fulfilled as soon as possible.