PLEASE HELP. I don\'t understand how step 8 would result in an \"unlimited suppl

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PLEASE HELP. I don't understand how step 8 would result in an "unlimited supply of DNA containing son of Rotund Gene". All they did was find cells that bind rotund protein. But all NORMAL mice cells bind rotund protein as well. For all we know from the information given, the genotype that causes the Son of Rotund phenotype could have nothing to do with that protein. And WHY FIBROBLASTS???? WHAT'S THE POINT OF THAT? Step one clearly discovers that rotund protein binds well to adipose tissue and hypothalamus

mixed together and ligated such that each plasmid will contain onNAd together will contain more than one. This is the plasmid cDNA Step : The digested cDNA and plasmids are Passage 6 (Questions 1-6) of obesity. These genes are often identifiedSepThe plasmid library is transferred into a human fibrobles of Rocund mice includes DNA from the exterior and express genes which have the proper Several genes have been identified that appear to play a role sferred into a human fibroblast ine. Transfection is a procedure in which eukaryoeg sie a the proper sigals Sep in the development of obesity. These genes are often identified obese mice n temperature and activity. When transfused with blood from normal mmal Rotund mice exhibit a momentary decrease in foode: The transfected cell population is grown in the preserno mouse molels for obesity, such as a pure-breeding strain of increRostund. The phenotype nsumption and body weight, and decreased body for transcripsion and translation. of radiolabeled cin and examined for individual cells that bind the radiol rotund protein. A fibroblast that binds rotund is identified and a clonal popalation of cells that bind rotund protein. intake. This information thata n suggests that a secreted factor exists that neomy ety, turning off the desire for food in normal animalsr when energy reserves in adipose tissue are sufficient. Stey The plasmid extracted from this population was found to be i kb larger than the plasmid in Figure 2. Bacteria were transfomed this new plasmid and grown in the presence of ampicillin, creating unimited supply of the DNA containing When the gene responsible for the Rotund phenotype was identified, it was found to encode a protein secreted from adipocytes in normal mice. This protein was termed the rotund protein. In Rotund mice, the gene for this protein contains a point mutation that results in a premature stop codon. Injection of purified wild-type rotund protein into Rotund mice induces a decrease in food intake, an increase in body temperature, an increase in activity, and a decrease over several weeks in the ratio of adipose tissue to skeletal muscle. Control injections lacking protein did not produce these changes. Also, injection of rotund proseid into normal ice does not alter weight or other parameters of mouse physiology, indicating that the weight loss in Rotund mice is not caused by toxicity of the protein to the animal. Son of Rotund gene. 400 150 Another mouse strain, termed Son of Rotund, has been identified which has a phenotype very similar to Rotund. The rotund gene sequence is normal in the Son of Rotund animals, and Son of Rotund mice do nol respond to injected wild-type rotund protein in any of the parameters measured. To identify the gene responsible for the Son of Rotund phenotype, the following experiment was performed. Experimental steps 8 normal mouse Se : Radiolabeled rotund protein is incubated with normal mouse remove unbosnd cells from different tissues. Cells are them w Binding of rotund protein to intact cells from normal procein before measurement of total bound radiclabeled protein. Both hypothalamus and adipase tissue showed strong binding of radiolabeled und protein, indieating the presence of a rotund receptor on these oells mice and mutant mice (see Figure 1). Endonuclease SV40 poly-A-addition Step 2: Total mRNA from the hypothalamus of a normal mouse is puried. This mRNA is reverse transcribed to produce double -stranded RSV-LTR (viral promoter) sequence Step 3: A plasmid is prepared with the struçture shown in Figure 2. This plasmid can replicate in both bacteria and human cell lines: additionally, it carries genes for antibiotic resistance. llin resistance Viral origin of replication Step 4: The plasmid is digested with an endonuclease which cuts at single site in the plasmid, as shown in Pigure 2. The cDNA is digested with the same endonuclease. a resistance E. coli origin of replication Figure 2 Structure of the rotund expression vector

Explanation / Answer

The fact that they have obtained a positive transformant with the desired construct enables them to obtain an unlimited supply of the desired protein. They can grow these transformants on selection media (Ex: Ampicillin containing media) and induce protein expression. They can inoculate the transformants inoculum into a huge volume of the media and obtain ample amounts of the protein by purification.

Each protein expression requires a specific host cell. For example, we can not express a biologically active human protein that harbors posttranslational modifications in prokaryotes. Similarly, some prokaryotic genes contain rare codons. Hence they can not be expressed in eukaryotic cells. In the given case, fibroblasts are chosen as the desired host cells for protein expression as they could support maximum yield.

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