Knockout mouse production assisted by Blm knockdown

摘要: To elucidate roles of any particular gene or genetic element in higher-order biological processes, a general method(s) for genome editing and creation such genome-modified species is essential. Therefore, the approach mouse embryonic stem cells (ESCs) ESC-based animal production commonly used. For targeting via homologous recombination (HR), vector possessing drug-resistant plus 5’ 3’ homology sequence arms introduced into cells. Although there well-established protocol many genes are already targeted by this method ESCs, efficiency varies at different target loci sometimes too low. Recently, other technologies as zinc finger nuclease (ZFN) [1, 2], TAL effector (TALEN) [3, 4] CRISPR/Cas9 [5, 6] systems have been developed. If specific highly competent can be designed, all these would work well mammals [7, 8]. The system newest systems, but it extremely useful. Unlike ZFN TALEN uses RNA guide molecule 20-nt specifies DNA site. preparation materials much easier simpler. Furthermore, if Cas9 mRNA delivered along with zygotes, genome-edited mice easily obtained. However, even system, some technical challenges still exist. An obvious one off-target mutagenesis risk due to restriction specificity. because homology-directed repair (HDR) less efficient mammals, replacement insertion mediated HDR inefficient zygote injection RNA, template construct generally not practical creating mice. For using standard vector, various trials applied improve frequencies HR [9]. Among them, knockdown Bloom syndrome gene, BLM, has shown enhance efficiencies human cell lines [10]. BLM encodes RecQ type helicase [11] plays role suppression [12]. yet investigated whether ESCs applicable knockout production. study, we multiple without Blm used ESC clones obtained chimeric germline transmission. For designed three siRNAs (siBlm1-3). At 48 h post transfection, amount was significantly decreased transfected independent (Fig. 1a). Western blot analysis also showed significant reduction protein specifically siRNA treatment 1b). siBlm-2 siBlm-3 induced higher than siBlm-1. selected combined them further experiments 1c). Fig. 1. Blm knocked down siRNAs. a) 20 nM each KY1.1. after level measured quantitative RT-PCR two primer sets. b) expression determined western blot. ... To validate how affects loci, namely, Prdm5 on chromosome 6, Prdm8 5 and, Arl14ep 2, pretreatment. expressed silent (not shown). We vectors 2a) [13]. Forty-eight hours before transfection vectors, part condition Fig. 1c. Then, were G418. More 200 colonies per screened proper targeting. As summarized Table 1, Prdm5, 8/214 (3.7%), 8/796 (1.0%) 14/240 (5.8%) knockdown, respectively. ones 29/232 (12.5%) 15/363 (4.1%) 32/240 (13.3%) Arl14ep. Thus, pretreatment pre-treatment enhanced fold activation enrichment 3.4 4.1 2.3 In another experiment targeting, control (siC-L) addition treated (Table 2). This time, low gave that siC-L (2.6 1.4 siC-L) suggesting effect non-specific. Fig. 2. Schematic diagram targeting: lox P (shaded triangle)-frt (open triangle)-PGK-Neo-frt site upstream downstream exon (Ex) Prdim8 P-frt-PGK-Neo-frt ... Table 1. Gene knockdown Table 2. Influence targeting Then, examined maintained pluripotency, especially transmission potential. Since sister chromatid exchanges (SCEs) increased [14,15,16,17,18,19], first checked stability. 3, performed karyotype (#12, #13 #27) parental ESC, KY1.1, control. examined, average number changed remained ~40. Table 3. Karyotype established knockdown Finally, created genes, 4, 2/3 4/4 generated > 80% judged coat color contribution. allele confirmed from those good (more gene). concluded does clear negative effects chromosomal stability potential clones. Table 4. Production knockdown In conclusion, provides benefit HR-mediated