343NUCLEAR LOCALIZATION SIGNAL AND CELL SYNCHRONY ENHANCES GENE TARGETING EFFICIENCY IN FETAL BOVINE FIBROBLASTS

Abstract

The use of primary somatic cells for nuclear transfer has facilitated the manipulation of the domestic animal genome via homologous recombination. Yet, the absolute frequency of homologous recombination (HR) in somatic cells is two orders of magnitude lower than in ES cells whereas frequencies of non-homologous end joining are higher. While a few loci have been targeted in somatic cells using enrichment strategies similar to those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells, and difficulties in cloning the cell after targeting. We present evidence that the use of a nuclear localization signal (nls) and S-phase cell cycle synchronization by thymidine block enhances targeting efficiency at the hypoxanthine phosphoribosyl transferase locus in primary fetal bovine fibroblasts. We designed two hypoxanthine phosphoribosyl transferase (HPRT)-targeting constructs, HPRT-DEx6 and HPRT-DEx6-nls. Both constructs have a 31-bp deletion and a PGK-puro insertion in exon 6 to ensure inactivation of the HPRT locus. Additionally, the HPRT-DEx6-nls construct contains a 180-bp cassette comprised of two 72-bp tandem repeats from SV40 enhancer known to act as nuclear localization signal. Diploid male cells that undergo targeted gene disruption at the single copy X-linked HPRT locus can be selected with 8-Azaguanine (8-AG) as HPRT cells are resistant to 8-AG; all transformants, random and targeted, can be selected in puromycin. Male primary bovine fibroblasts were electroporated with linearized targeting constructs, and plated in media containing puromycin or puromycin plus 8AG. All experiments were done in triplicate and data were analyzed by two-way ANOVA with NLS and cell synchrony as the two factors. Significance was set at P<0.01. While the total number of insertions (random plus targeted) with both constructs were equivalent, the HPRT-DEx6-nls construct produced a significantly higher number of targeted colonies (1–2 8AG-resistant colonies per 9.5×106 cells) than HPRT-DEx6 where no targeted events were seen. Cells were synchronized in the S-phase of the cell cycle by a 2mM thymidine treatment for 24 hours and electroporated with the linearized targeting constructs. Compared to non-synchronous cells, the total number of insertions (random plus targeted) was reduced by 59-fold in constructs with or without nls (P<0.01), while targeted insertions increased 6-fold in the HPRT-DEx6-nls construct, from an average of 10 per million cells without nls to 7.6 per 10 million with nls (P<0.01). All 8AG-resistant colonies were verified by Long Range-PCR, and PCR products confirmed by end sequencing. This finding has important implications for targeting in somatic cells, as with a drastic reduction in the number of random insertions, and increased targeting due to the presence of the nls, identification of a targeted colony is greatly facilitated even in cases where no enrichment protocols are available.