Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.
Amphotericin B-induced synovitis of the left tarsocrural joint was used to create a grade 3 of 4 lameness in 11 horses. Caudal epidural catheters were placed and advanced to the lumbosacral region. Baseline heart and respiratory rates were recorded and horses were videotaped at a walk and trot. Morphine sulphate (0.2 mg/kg) and detomidine hydrochloride (30 micrograms/kg) were administered to treated horses (n = 8) through the epidural catheter; an equivalent volume of physiologic saline solution was administered to control horses (n = 3) through the catheter. At hourly intervals after epidural injection for a total of 6 hours, heart and respiratory rates were recorded, and horses were videotaped walking and trotting. At the end of the observation period, video recordings were scrambled onto a master videotape. Lamenesses were scored by three investigators unaware of group assignment or treatment time. Lameness scores, heart rates, and respiratory rates were compared between groups using repeated measures analysis of variance. There was a significant decrease in lameness score after treatment with epidural morphine and detomidine (P = .0003); average lameness scores of treated horses were less than grade 1 at each hourly observation for 6 hours after drug administration. Early in the observation period, heart rates significantly increased in control horses and decreased in treated horses (P = .03). A similar trend occurred for respiratory rates (P = .07). Results of this study demonstrate that epidural administration of a combination of morphine and detomidine is capable of providing profound hindlimb analgesia in horses.
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