The transformed secretory gland cells provide long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein. Description This application is a continuation of U. The work carried out by Avery et al. The insertion of new genetic material into cells in order to permanently affect the genetic makeup of the cells is the methodology now generally referred to as gene therapy.
Rubber-Maid or Tupperware tray or equivalent sealable tray Parafilm cut into 22 mm squares g weights 3. Fixatives and solutions 5X formaldehyde stock. Prepare a fresh solution of 0. Warm to disperse the Triton X and use within 1 hour.
Prepare a fresh solution of 1. Prepare a solution of 1 ml lactic acid, 2 ml dH2O, and 3 ml acetic acid. Polytene chromosome squash preparation: Rinse larvae with water and transfer to PBS in a tissue culture dish for dissection. Separate the salivary glands from the brain and eye-antennal discs, and dissect away the fat body and any other associated tissues from the glands.
Add microliters of Fixative 1 to the first well of a two well depression slide and microliters of Fixative 2 to well two. Transfer one pair of salivary glands at a time to Fixative 1 in well one and incubate for the amount of time required for the target epitope, usually about minutes. Using forceps transfer the salivary glands to Fixative 2 in well 2 and incubate for two minutes.
Gently lower a polylysine-coated microscope slide onto the coverslip and, without applying vertical pressure, pick up the coverslip so the glands are between the slide and the coverslip. Immediately facilitate cell lysis and chromosomal spreading by carefully grasping the coverslip with forceps on one edge and gently moving it slightly back and forth, trying to minimize any vertical pressure on the tissue.
Alternatively use the eraser side of a pencil or even a fingertip to gently move the coverslip back and forth. Note that any delay in moving the coverslip back and forth is likely to diminish spreading of the chromosomal arms as they will tend to become more rigid upon exposure to the Lactoacetic acid solution.
Clouding of the solution is often a good indication of cell separation. Gently tapping the coverslip obliquely to avoid vertical pressure with the eraser side of a pencil a few times may also assist in chromosomal spreading. Immediately examine the tissue under a phase contrast microscope with a 20 or 40X objective to determine whether the chromosomes are well-spread.
When the chromosomal spreading is sufficient, set the slide with the coverslip side down on a stack of clean Kim-wipes. Place a second Kim-wipe on top and flatten chromosomes by placing your thumb over where the coverslip is positioned and pressing firmly, avoiding any horizontal movement of the coverslip that would shear the chromosomes.
Examine the slide again under the microscope to determine if the preparation is suitable for the intended purpose. If chromosomes have moved significantly upon squashing, use less volume of Lactoacetic acid solution in your subsequent preparations.
Place g weights upon the coverslip of the slides.
Fill a small Dewar e.Example of a High Quality Squash of identified on the basis of their characteristic shapes and sizes.
5 m Lab 4A. Preparation of Drosophila Polytene Chromosome Squashes A response is required for each item marked: (#__). Your grade for the lab 4 report (lab 4A and 4B chromosomes from the Drosophila larval salivary gland for his . In this regard, the promise of DNA barcoding (Rivera and Currie, ), proteomic (Cupp and Cupp ; Cupp, et al., ) and genomic (Procunier WS, et al., ) approaches that target the functionally important salivary gland may also be instructive for understanding evolutionary biology and hierarchal classification of this family.
Polytene chromosome. for example, in Chironomus and are called Balbiani rings after their discoverer. Puffs are reversible modifications of polytene chromosomes; (BR) in principle are giant puffs in the polytene chromosomes of the salivary gland cells of larvae of Chironomus tentans, as first described in by Balbiani.
They are. A native salivary gland chromosome IV that has been isolated at pH does not appear any different than a chromosome IV in a squash prepara- tion. All of the prominent landmarks such as Balbiani rings and genetically determined constrictions can be found in their normal sites and configurations.
Salivary Gland Larval D. Melanogaster Polytene Formed by Endomitosis Essay Sample. The rearing of D. melanogaster under specific conditions and the experimental extraction of the salivary gland chromosome permits the cytological investigation of the structure, formation and functioning of the polytene (giant) chromosomes and genes during the larval development.
Salivary Gland Chromosome Preparation Essay. Introduction: Microscopic, threadlike part of the cell and a structured DNA which carries hereditary information in the form of genes is a chromosome - Salivary Gland Chromosome Preparation Essay introduction. Endomitosis is mitosis without nuclear or .