An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios

In silico modeling indicates the development of HIV-1 resistance to multiple shRNA gene therapy differs to standard antiretroviral therapy

<p>Abstract</p> <p>Background</p> <p>Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus.</p> <p>Results</p> <p>We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral <it>status quo</it>.</p> <p>Conclusions</p> <p>The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy.</p

Selected RE recognition sites with compatible cohesive ends.

<p>Four pairs of RE recognition sites with compatible cohesive ends were suitable for the plasmid used in this study. The 4 pairs were divided into 2 ‘core’ sets so that the enzymes with the most similar buffer requirements were grouped together, based on the % activity of each enzyme in the 4 different New England Biolabs buffers plus standard PCR buffer (catalog & Technical Reference, 2007–08). <b>^*</b><i>Bsi</i> WI was optimally active at 55°C and 50 % active at 37°C.</p

Site allocation and compatibilities.

<p>The MCS inserted into the recipient plasmid was assembled from <i>Age</i> I, <i>Spe</i> I, <i>Nhe</i> I, <i>Blp</i> I, <i>Bsr</i> GI, <i>Mlu</i> I, <i>Asc</i> I, <i>Sma</i> I, <i>Pac</i> I, <i>Acl</i> I, and <i>Dra</i> III. PCR donor fragments made with core 2 primers (A) are digested with <i>Mlu</i> I and <i>Asi</i> SI and inserted into <i>Asc</i> I and <i>Pac</i> I (B). Previous insertions (C) are excised with <i>Mlu</i> I and <i>Pac</i> I and sub-cloned into another vector prepared either previously or in parallel with (C) and opened with <i>Asc</i> I and <i>Pac</i> I (D). Entire cloning regions (all cores and inserts) can be shuttled between vectors (E–F) using the external shuttling sites, <i>Age</i> I and <i>Acl</i> I or <i>Dra</i> III. * <i>Blp</i> I and <i>Sma</i> I were included as spacers to distance the two sites to be double digested for receiving inserts.</p

Sub-cloning sub-combinations is the most time-efficient way to build any combination greater than four.

<p>(A) The sub-cloning protocol can hasten the completion of large projects through parallel lines of construction in multiple vectors that are progressively joined together. For example, a combination of 11 could be made in 11 consecutive rounds of sequential PCR insertions, or more (time) efficiently in 5 rounds of sub-cloning sub-combinations. (B) The minimal number of rounds required to complete any given combination (n) was found by: ⌈log₂(n)⌉+1 (when starting from scratch). Calculations show that sub-cloning is the most time-efficient construction strategy for any combination of four or more insertions (solutions shown for all combinations up to 65).</p

Combinations up to seven built from PCR donor fragments.

<p>Seven different hairpin expression cassettes were amplified with the core 2 primers and inserted both individually and sequentially to create combinations of increasing number up to seven in the recipient plasmid. Insertions were confirmed by PCR screening using primers that flanked the MCS region. Each insertion added ∼270 bp to the size of the previous recipient plasmid.</p

Pfu was used to efficiently amplify repeated sequences by PCR.

<p>(A) Taq-based PCR was unsuitable for screening multiple insertions as it produced strong intermediate-sized products and weak specific-products when amplifying templates containing 1 to 7 hairpin expression cassettes. (B) A series of approximately equivalent sized plasmids with non-structured (& non-repeated) inserts was successfully amplified with Taq. (C) Taq was unsuitable for amplifying templates containing 1 to 7 promoter-only cassettes (no hairpin sequences). (D) Several different polymerases (Phusion, Dynazyme EXT, Dynazyme II, Immolase and Pfu) were tested with the promoter-only series of vectors using the manufacturers recommended starting conditions.</p

An infinitely expandable cloning strategy.

<p>The PCR generated donor fragment (A) is digested with ‘A’ and ‘b’ enzymes and ligated to the recipient vector (B) opened up with ‘a’ and ‘B’ enzymes destroying the original ‘A’, ‘b’, ‘a’, and ‘B’ sites in the process. The newly created vector (C) has the ‘a’ and ‘B’ sites reconstituted. Further insertions stack after each at the expansion point (XP), and each insertion leaves two non-functional digestion or ligation remnants, the downstream ones stacking together at a single point (RS). Sub-cloned donor fragments (C) from previously constructed vectors are excised with ‘A’ and ‘B’ enzymes and ligated to a recipient vector (D) opened up with the ‘a’ and ‘B’ sites. In this example the recipient vector (2) already has one inserted cassette, thus making a new vector with a total of two cassettes (E).</p