Improved methods for the generation of human gene knockout and knockin cell lines

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells

Cdk2 Is Required for p53-Independent G2/M Checkpoint Control

The activation of phase-specific cyclin-dependent kinases (Cdks) is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G2/M checkpoint activation

Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

<div><p>The adenoviruses are essential tools for basic research and therapeutic development. Robust methods for the generation of mutant and recombinant viruses are crucial for these diverse applications. Here we describe a simple plasmid-based method that permits highly efficient modification of the adenoviral genome and rapid production of high-titer virus stocks. The 36-kilobase genome of adenovirus serotype 5 was divided into seven tractable blocks that could be individually modified in a single step and reassembled <i>in vitro</i>. Because the system is composed of compact modules, modifications at different loci can be readily recombined. Viral assemblies were delivered to packaging cells by electroporation, a strategy that consistently resulted in the <i>de novo</i> production of 10⁸ infectious units in 3–5 days. In principle, a similar strategy could be applied to any other adenovirus serotype or to other double-strand DNA viruses.</p></div

Seamless ligation of adjacent genomic blocks.

<p>The free ends created by BstBI are initially processed by the heat-labile T5 exonuclease, creating an extended 3’ overhang at each end. When complementary strands anneal, the ends are trimmed, filled in and ligated. The thermostable BstBI enzyme remains active during the assembly process and thus prevents re-ligation of the cut ends. The compatible ends of Blocks 1 and 2 are shown; the other junctions are simultaneously processed in the same manner.</p

Overview of the Adenobuilder system.

<p>Segments of the Ad5 genome were individually amplified and cloned into high-copy plasmids. Each of the five central “building blocks” contains unique sequences at either end (represented by distinct colors) that create a 15–20 bp overlap with the respective neighboring block. Each block is flanked by recognition sites for the restriction enzyme BstBI. Following digestion, the unpurified plasmid fragments are directly added to an isothermal assembly reaction and then delivered to packaging cells via electroporation. This system thus facilitates the generation of infectious adenovirus particles from plasmids in three steps that can be carried out in under two hours.</p

Two-step derivation of mutations in E1B and E4.

<p><b>A.</b> The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. <b>B.</b> Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. <b>C.</b> hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. <b>D.</b> hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.</p

Partitioning of the Ad5 genome.

<p>The cloned blocks 1–7 are aligned to a map of the Ad5 genome. Protein-coding genes are indicated in red, with arrows indicating the strand that is transcribed. The inverted terminal repeats (ITRs), which define the left and right ends of the adenovirus episome, and virus-associated RNAs (VA RNA) are shown. The early and late transcriptional units are marked by green and black arrows, respectively.</p

Modifications to blocks 1, 6 and 7 facilitate the incorporation of large transgenic elements into recombinant Ad5 vectors.

<p><b>A</b>, DNA cassettes up to 3 kb in size can be directly cloned into a polylinker in the modified block pAd5-B1ΔE1-MCS. Still larger transgenic DNAs can be incorporated into assembled viruses with the use of plasmids pAd5-B6ΔE3 and/or pAd5-B7ΔE4. <b>B</b>, successively larger transgenes can be introduced with the deletion of E1, E3 and E4. Used together, these three modified blocks thus allow the customized assembly of recombinant viruses capable of delivering transgenic elements up to 8.5 kb in size.</p