Creating the targeting constructs of Scrt1 and Scrt2 to study their function in the dorsal spinal cord Hreem Dave1, Dr. Wenlin Yuan and Dr. Zhou-Feng Chen2, Biology Department, Washington University, St. Louis, MO1, Department of Anesthesiology, Washington University School of Medicine2 The dorsal spinal cord is the major region for integrating and relaying sensory information from our skin to our brain. The majority of neurons lie along the dorsoventral axis and provide the anatomical framework that underlies the physiological function of sensory and motor neurons. Therefore, an injured dorsal spinal cord could lead to loss of pain sensation in our body. In order to develop an effective treatment for spinal cord injuries, more about the maturation of the spinal cord must be studied. The main problem with tracing the development of the dorsal spinal cord is that only a relatively small number of molecules that specifically control cell lineages, migration pathways, and axonal branching have been identified. A partial sequence of a new gene with five zinc fingers was identified through a homology search of the mouse genome. The new gene, Scratch2 (Scrt2), is homologous to Scratch1 (Scrt1) and belongs to the Snail superfamily. The Scratch family genes are the representations of the Snail superfamily with significant expression in the nervous system. The exact role of Scrt1 and Scrt2 in the dorsal spinal cord has not been determined but it is known that they are expressed in the developing dorsal spinal cord and share an overlapping expression pattern. These genes may play an essential role in migration and differentiation of dorsal horn neurons. One way to determine the role of Scrt1 and Scrt2 is to conduct loss-of-function studies with mice. In order to create these knockout mice, a gene targeting construct must be created. The overall process consists of cloning and amplifying the 5’ and 3’ homologous regions. A series of PCRs, restriction enzyme digests, gel purifications, ligations, and transformations were carried out in an attempt to clone a section of the 5’ homologous region of Scrt1. First, a restriction enzyme, EcoRV was used to cut the plasmid vector, BSKS (Blue Script), to generate a sticky end vector. Afterwards, an enzyme, Klenov polymerase was used to append phosphates to the sticky end so they became blunt ends. Meanwhile, a blunt end insert was created through PCR of the 5’ homologous region. In order to allow the insert to attach to the vector, a ligation was prepared and left overnight at room temperature. Afterwards, the ligation was plated out onto LB/+amp plates and the vectors that seemed to have ligated correctly were selected. If the colonies tested positive for the correct insert, then it was sent off to the DNA sequencing center. Finally, if the sequencing matched the 5’ homologus region that was being cloned, the cloning would be complete and a new part of the construct would be finished. The complete targeting construct will create knock out mice and this will lead to a better understanding of the role of Scrt1 and Scrt2 in the development of the dorsal spinal cord. Scholar Index Next   Natural Sciences Learning Center Washington University - Biology All contents copyright © 2003 Email comments to nslc webmanager