Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.

[Clinical verification of serum procalcitonin measurement in sepsis].

To evaluate the clinical application of measuring procalcitonin (PCT) level for diagnosis of bacterial sepsis in patients with and without systemic inflammatory response syndrome(SIRS), we studied the relationship between blood culture (BC) and serum PCT level in clinical 207 cases. In addition, we evaluated the time courses of PCT and other inflammatory markers: tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), E-selectin, WBC count and C-reactive protein (CRP) in 5 bacterial septic patients with SIRS. Serum PCT showed sensitivity of 41% and specificity of 61%, while BC showed specificity of 88%. In 27 BC-positive cases, serum PCT was significantly elevated in gram-negative bacterial sepsis. We observed 11 cases with BC(+) and serum PCT below 0.5 ng/ml. Major causes of these discrepancies were probably due to gram-positive bacterial infection, local bacterial infection or pretreatment with broad-spectrum antibiotics. In contrast, 10 cases with BC(-) and serum PCT over 10 ng/ml were presumably due to some cytokine elevation caused by virus infection or collagen diseases. In 5 cases studied for inflammatory markers, TNF-alpha level elevated earlier than the others and followed by PCT, IL-6, WBC, CRP, and E-selectin. It was suggested that the measurement of serum procalcitonin in septic patients is clinically useful marker to diagnose gram-negative bacterial septic patients with SIRS.