Lentiviral vectors and plasmids FAQ

• What generation are your lentiviral vectors? Why does generation matter?

  Imanis lentiviral vectors and transfer plasmids are second generation. Generation matters when considering which packaging system to use to produce lentiviral vector particles from the transfer plasmids. Second generation lentiviral vector systems use fewer plasmids than third generation systems in order to produce lentiviral vector particles. Also, some institutes and countries have different safety regulations for second versus third generation lentiviral vectors.

  To increase biosafety, all of our lentiviral vectors are self-inactivating (SIN) vectors, in which the viral enhancer and promoter have been deleted. Transcription inactivation of the LTR in the SIN provirus prevents mobilization by replication competent viruses and enables regulated expression of the genes from the internal promoters without cis-acting effects of the LTR.

• What is replication competent lentivirus (RCL)?

  Replication competent lentiviruses (RCL) are virus particles capable of infecting cells and replicating to produce additional infectious particles.

  The existence of RCL in stable cell lines generated by lentiviral vector transduction is a safety concern. As such lentiviral vectors have been engineered to significantly reduce the likelihood of RCL production. These engineered lentiviral vectors have an excellent safety record, as there are no known reports of actual RCL production.

  Although RCL remains a theoretical concern, most institutes have specific requirements related to testing transduced cells for RCL. A p24 ELISA assay is the most commonly used test to demonstrate the absence of RCL in a sample of transduced cells. Imanis offers p24 ELISA RCL testing for lentivirus; visit our Sample Analysis page for more information.

• What is the source of the transgenes?

  Our transgenes come from a variety of sources. Check out the table below for detailed information about the source of our transgenes.

  Transgene	Accession #	Gene Source	Description
  Luc2	AY738222	Photinus pyralis	Codon optimized firefly luciferase with brighter signal1.
  hNIS	U66088	Homo sapiens	Human sodium iodide symporter; mediates iodide uptake2.
  hNISplus	Mutant of U66088	Homo sapiens	Genetically modified hNIS with enhanced radiotracer uptake.
  mNIS	AF235001	Mus musculus	Murine (mouse) sodium iodide symporter; mediates iodide uptake3.
  rNIS	U60282.1	Rattus norvegicus	Rat sodium iodide symporter; mediates iodide uptake4.
  pigNIS	NM214410	Sus scrofa	Pig sodium iodide symporter; mediates iodide uptake5.
  RhNIS	N/A*	Rhesus macaque	Rhesus sodium iodide symporter; mediates iodide uptake.
  dNIS	XM_541946	Canis lupus	Dog sodium iodide symporter; mediates iodide uptake6.
  IRES	M81861 (nt 260 to 848)	EMCV	Internal ribosome entry site; RNA element that mediates internal translation initiation7.
  eGFP	AAB02572.1	Variant of GFP from Aequorea victoria	Enhanced GFP; mutant of GFP with a 100-fold increase in fluorescent signal8.
  iRFP	JN247409	Rhodopseudomonas palustris	Near-infrared fluorescent protein; red-shifted fluorescent protein with an excitation/emission of 690/713 nm9.
  dsRed	AB212907	Anopheles gambiae	Red fluorescent protein variant with an excitation/emission of 558/583 nm10.
  DRD2	NM_016574.3	Homo Sapiens	Dopamine receptor D2; endogenously expressed in the brain11.
  hNET	NM_001172501.1	Homo Sapiens	Human norepinephrine transporter; endogenously expressed by noradrenergic neurons12.
  SSTR2	AY236542.1	Homo Sapiens	Somatostatin receptor 2; endogenously expressed in cerebrum and kidneys13.
  HSV-TK	JQ352282.1	HSV	Human herpesvirus 1 thymidine kinase (UL23)14.
  hTYR	M27160.1	Homo sapiens	Tyrosinase; converts tyrosine to brown-pigmented melanin15.

  *Sequence derived from a cDNA library. Email support@imanislife.com for more information.
  ¹ Promega
  ² Smanik et al. Biochem Biophys Res Commun. 1996. 226(2):339-45
  ³ Pinke et al. Thyroid. 2001. 11(10):935-9
  ⁴ Dai et al. Nature. 1996. 379(6564):458-60
  ⁵ Selmi-Ruby et al. Endocrinology. 2003. 144(3):1074-85
  ⁶ Uyttersprot et al. Mol Cell Endocrinol. 1997. 131(2):195-203
  ⁷ Gurtu et al. Biochem Biophys Res Commun. 1996. 229(1):295-8
  ⁸ Cormack et al. Gene. 1996. 173(1):33-8
  ⁹ Filonov et al. Nat Biotechnol. 2011. 29(8):757-61
  ¹⁰ Baird et al. Proc Natl Acad Sci USA. 2000. 97(22):11984-9
  ¹¹ Dearry et al. Cell Mol Neurobiol. 1991. 11(5):437-53
  ¹² Pacholczyk et al. Nature. 1991. 350(6316):350-4
  ¹³ Parry et al. Mol Imaging. 2007. 6(1):56-67
  ¹⁴ Koehne et al. Nat Biotechnol. 2003. 21(4):405-13
  ¹⁵ Takeda et al. Biochem Biophys Res Commun. 1989. 162(3):984-90

• Can I get the complete sequence of a plasmid?

  We will happily provide full sequences for any plasmids upon purchase.

• What packaging system should I use with your plasmids to generate lentivirus?

  Imanis lentiviral vector transfer plasmids are meant to be used in a 3 plasmid second generation packaging system, which includes the following:

  1. A lentiviral transfer plasmid (from Imanis): encodes transgene and promoter
  2. A packaging plasmid: encode Gag, Pol, Rev, and Tat (Tat is essential for second generation packaging).
  3. A envelope plasmid: encoding the envelope protein (usually VSV-G).

  Our transfer plasmids cannot be used with third generation packaging systems.

• What is the difference between a p24 and qPCR titer?

  A p24 ELISA detects the lentiviral p24 Gag protein. It is not specific for function lentiviral vector particles; it measures free p24, p24 associated with non-functional vector particles, and p24 in functional vector particles. Thus, traditional p24 ELISA titrations overestimate the functional titer. And because the ratio of free p24, p24 associated with non-functional vector particles, and p24 associated with functional vector particles can vary greatly between each lentiviral vector lot, the titers are inherently inaccurate.

  In contrast, qPCR titration quantifies the number of integrated lentiviral genome copies per cell in a population of transduced cells. Thus, qPCR titers yield a functional and more accurate titer, though they often appear lower than p24 titers. All Imanis lentiviral vectors are titered by qPCR.

• How much of your lentivirus should I use for a transduction?

  The optimal amount of lentivirus for transduction is cell-type dependent (e.g. primary cells require more virus than established cell lines). We recommend determining the optimal MOI for transduction by plating several wells of the target cells and infecting with increasing MOIs (e.g. 1, 3, 10, and 30*).

  *For more information about calculating MOI, please see Cell Biology Protocols.

• Do I need biosafety approval to use your lentiviral vectors?

  Work with lentiviral vectors should be carried out using BSL2 practices. However, institutional requirements vary, so check with your institution’s biosafety officer to confirm the requirements for lentiviral vector use.

  Typically, lentiviral vector work in vivo must have both IBC and IACUC approval, and in vitro work with lentiviral vectors should be performed under BSL-2 or BSL-2+ containment practices.

• Do you have a transduction protocol?

  Yes! Our transduction protocols are listed under the “Transduction” tab on our lentiviral vector product pages.

• How much does the lentiviral vector titer drop with freeze-thaw cycles?

  The titer of lentiviral vectors will drop with freeze-thaw cycles. In-house studies have shown that the titer drops approximately 50% with each freeze-thaw cycle, though this number will vary. We recommend avoiding freeze-thaw cycles. If it is necessary to re-freeze lentiviral vectors for future use, the lentiviral vectors should be aliquoted into single-use aliquots.