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RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.
RNA interference (RNAi) and the green fluorescent protein (GFP) are two methodological advances that have transitioned so rapidly and effectively from discovery to widespread usefulness that they now seem indispensable to basic biomedical scientists. Their utility was recognized by Nobel Prizes awarded in 2006 and 2008, respectively. Though each of their core biological mechanisms evolved to support intricate, phenotypically striking processes – whole organism RNA silencing effects in plants and worms and the bioluminescence of ocean invertebrates – a hallmark of both technologies is relative simplicity in practice. The method reviewed here combines them with an accessible gene transfer technology that is particularly suited to facile use by HIV researchers. We describe how to establish stable, stringent RNAi using lentiviral vector transduction of short hairpin RNAs (shRNAs) followed by optional sorting for co-encoded GFP or one of its numerous spectral variants. The techniques described enable well controlled, phenotype revealing knockdowns in human T cell lines and other targets. They also allow the researcher to modulate the degree of knockdown.
Numerous in-depth reviews of RNAi and lentiviral vectors are available and other groups have used shRNA expression similarly to good effect. In keeping with the theme of this volume, we draw on our own experience to concentrate on providing practical advice to researchers studying early events in HIV-1 replication, including integration. However, the methods are easily adapted to other fields. We also discuss ways to intensify RNAi when needed, as it was for establishing the role of lens epithelium-derived growth factor (LEDGF/p75) in HIV-1 replication . Similar knockdown stringency may also be useful for studying other viral dependency factors with roles in early events. Intensification can be especially significant for those factors that are spatially concentrated in a functionally critical way, e.g., by chromatin association . We focus on CD4-positive T cell lines, explain proper controls to validate RNAi specificity, and also detail how to assess HIV-1 and control retrovirus phenotypes after knockdown.
shRNAs mimic natural RNAi in ways that synthetic siRNA oligonuceotides do not . RNAi is initiated normally by long double-stranded RNA (dsRNA), which serves as a substrate-trigger for Dicer, a ribonuclease III that cleaves dsRNA into 21–23 nucleotide small interfering RNAs (siRNAs) . siRNAs function within a multi-protein complex called the RNA-induced silencing complex (RISC) . RISC contains helicase activity necessary to free the antisense guide strand to form the RNA-protein complex siRISC. The guide strand cooperates with the endonuclease Argonaute2 to guide sequence-specific degradation of mRNA 10–11 bases upstream of the 5’ end of the guide strand, ultimately leading to gene silencing [4–6]. While siRNAs can be administered directly and incorporate into RISC without any cellular modification, Dicer-generated siRNAs are generally more potent than their synthetic counterparts . shRNAs are transcribed from sense and antisense DNA sequences, generally 19–29 nucleotides in length, which are connected by a short intervening linker . Thus an advantage of shRNAs is that their stem-and-loop secondary structure generates siRNAs in a Dicer-dependent manner . They also provide the obvious advantage of allowing stable expression from DNA constructs. The short length of shRNAs also minimizes potential activation of mammalian double stranded RNA-dependent protein kinase R (PKR) [9, 10], limiting broad off-target effects. shRNA expression is often carried out with RNA polymerase III (pol III) promoters (e.g. H1 and U6), which encode specific initiation and termination sequences that generate the optimal 2-nt (uracil) 3’ overhangs. However, pol II promoters can also be used. For a review of this issue, see .
In situations where small residua of the target protein retain significant biological activity, as exemplified by LEDGF/p75, a good approach is a gene-level knockout in mice. Human cell knockdowns and mouse cell knockouts were both instrumental for understanding the function of LEDGF/p75 in the HIV-1 life cycle [1, 11, 12]. However, murine cells have diverse, complex blocks to HIV-1 replication, which prevents testing of the full life cycle. Mouse embryonic fibroblasts are also, for a variety of experimental reasons, more challenging to adapt to controlled HIV experiments than are human primary and immortalized human CD4+ T cells. Primate cell knockouts seem logical solutions, but remain very difficult in practice. Intensified RNAi in human cells is therefore often a worthwhile alternative.
We review our approach, intensified lentiviral vector RNAi (ilvRNAi) in human T cells, in which the capacity of lentiviral vectors for effective stable transduction of both dividing and nondividing cells is combined when needed with sorting for co-transduced fluorescent proteins to achieve knockdowns of graded intensity. Properly executed, the complete method includes control shRNA transductions and re-expression with RNAiresistant cDNAs. Although lentiviral vector systems have now been derived from primate and non-primate lentiviruses (reviewed in  and ), the higher transduction efficiency of HIV-1 vectors in most human CD4+ T cell lines and primary lymphocytes (in our hands up to 10–20 fold per unit of particle reverse transcriptase activity) makes them preferred for these targets. Because the transfer vectors we use are U3-deleted, deriving cell lines with HIV-1-based vector transduction does not interfere with subsequent HIV-1 infection studies: the vectors are not rescued by replicating HIV-1 and the missing 400 nt can be used to anchor 2-long terminal repeat (LTR) circle PCR and Alu-PCR for HIV-1 challenge virus integrants.
The following protocols can be adapted by any laboratory experienced in molecular biology and mammalian tissue culture. Figure 1 illustrates the basic stages of developing and using ilvRNAi, which include:
Our protocols will highlight three points: 1) achieving deep knockdown to eliminate residual protein expression; 2) assaying quantifiable, relevant endpoints to validate knockdown phenotype; and 3) attention to the right controls to verify RNAi specificity and prove gene function.
Several issues are relevant when designing an shRNA construct: the promoter, the target sequence, and the shRNA construct sequence. pol III promoters, e.g., U6 and H1, are useful because of their precise start and termination sequences. We have used both in human T cell lines.
Active siRNAs are generally 21-nt long with 3’ dinucleotide overhangs and 19-nt complementarity . A number of algorithms are available for choosing optimal 19-nt targets [17, 18]. The Ambion RNAi interference web resource is helpful (http://www.ambion.com). In general, it is reliably useful to choose 19-nt target sequences that begin with AA, contain less than 50% GC content, and do not contain homo-repeats greater than 3 nucleotides. Those 19-mer sequences that have been functionally validated with siRNA oligonucleotides are likely to be effective when used as shRNAs, and a review of previously published siRNAs or shRNAs is a sensible way to start. Sequences longer than 21-nt can be used, and may actually be more efficient Dicer substrates [7, 8], but there is a risk of confounding PKR induction [9, 10]. We have found both 21-mer and longer (27-mer) sequences to be effective. The chosen sense sequence is then linked to the antisense sequence with an intervening loop of variable length. We use the 9-nt sequence TTCAAGAGA  but most evidence suggests this is not critical. For a control shRNA target we have used actgccgttgttataggtg, which in our and others (http://www.ambion.com) hands has been phenotypically neutral even at high MOIs. Figure 2 depicts transfer vector TSINcherryU6, which we have modified from the lentiviral vector of Llano et al.  in two ways: (1) introducing unique restriction sites that enable shRNA insert cloning in a single step; (2) making the human U6 start resemble the native promoter more closely. This is as effective as the original shRNA transfer vector when tested head to head for LEDGF/p75 knockdown and HIV-1 resistant phenotype (our unpublished data).
Sense and antisense oligonucleotides for the shRNA insert need to be synthesized so they contain the pol III termination sequence (TTTTTT) and when annealed yield adaptors at their termini that match the vector. In TSINcherryU6 (Fig. 2), MluI and SalI are positioned for this purpose.
Lentiviral vector particle production is performed by co-transfection of the shRNA transfer vector with an envelope glycoprotein expression plasmid and a packaging plasmid. Examples of the latter include pCMVΔR8.9, which encodes gag/pol, tat and rev . Alternative 4-plasmid systems supply Rev from a separate plasmid, e.g., the pLP1 + pLP2 combination (Invitrogen) or pCHGP and pCMV-Rev . Rhabdoviral (VSV-G) envelope glycoprotein pseudotyping expands vector tropism and enhances vector titers and stability .
If vector production was successful, unconcentrated vector titers should be at least 1 × 106 TU/ml. Concentrated vectors are typically at least 1 × 108 TU/ml depending on the starting and final resuspension volumes. Generally only a fraction of a microliter of a vector with 108 TU/ml is required to fully transduce 50,000 cells. Keep this in mind when titering vector to ensure samples are in the linear range for titer calculation.
Generating stable cell lines requires transducing target cells with vector at increasing MOIs, cryopreserving and analyzing cells for a relevant knockdown phenotype. If desired, enrichment by FACS can be performed. Differential transduction efficiency and sorting allow graded RNAi intensity.
Potential off-target effects make proper controls necessary for correct interpretation of RNAi experiments. A consensus gold standard is target-protein re-expression and pre-knockdown phenotype rescue [25, 26]. This step also controls for possible effects of target protein over-expression, as does parallel over-expression in cells lacking the knockdown. We have used gamma-retroviral (MLV) vectors for re-expression, although stable plasmid transfection is also an option . For obvious reasons, lentiviral vectors are poor choices in the presence of a knockdown of a dependency factor that acts between lentiviral entry and integration (e.g., LEDGF/p75).
This protocol allows researchers familiar with basic molecular biology and cell culture to rapidly and effectively perform transient or stable knockdowns with lentiviral vectors. One-step construction of ilvRNAi transfer vectors allows rapid progression from thinking about a gene target to analyzing knockdown results. RNAi intensification by sorting for stably high-expressing populations is readily performed though is not always necessary. For HIV researchers, the ability to effectively knock down individual genes in CD4+ T cells allows rapid analysis of new targets. Indeed, high throughput siRNA library-based screens [31, 32] are now identifying numerous candidate HIV-1 dependency factors that can be subjected to confirmatory studies by focused methods such as ilvRNAi. Details of these large scale transcriptome studies also illustrate how the degree of achievable intensification can matter. For example, LEDGF/p75 was originally brought to the attention of HIV researchers by an affinity-based proteomics screen for integrase interactors ; see  and  for reviews. It’s potency as a viral cofactor led to a prediction that it would likely be missed by broad RNAi screens , which turned out to be the case . The second large scale screen also found minimal (3–8%) inhibition of single cycle HIV-1 infection with pooled LEDGF/p75 siRNAs , which prospectively might not be regarded as an actionable hit. The point is that by virtue of their breadth, such screens cannot generally approach the RNAi intensification needed to identify and confirm factors that retain viral cofactor activity at a small fraction of their endogenous levels.
We thank I. Kemler and N. Gaznick for assistance with pTSINcherryU6 construction
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iThese values can vary. Early lentiviral vector protocols emphasized quite large DNA amounts in transfections, but this is not necessarily required. We have used either 3 or 10 µg of transfer vector depending on whether we are using a 3:3:1 or 10:10:3.3 µg ratio (transfer vector to packaging to VSV-G expression plasmids). The lower amount works well for feline immunodeficiency virus (FIV)-based vectors, but we have generally used the larger amount for HIV-1 vectors. In Nunc Cell Factories, we have generally used a 3:3:1 ratio for all types of vectors to conserve DNA.
iiWe generally collect at 48 h after washing off the transfection mix. An alternative is to collect at both 24 and 48 h for both HIV and MLV vectors and pool the supernatants, but this yields twice the volume to concentrate.