The Sal-like 1 gene (Sall1) plays a role in kidney development. Point mutations in the gene result in abnormalities in the kidney and mice lacking the Sall1 gene show agenesis (absence) of the kidneys. As a preliminary step towards the generation of a human kidney in a pig, researchers developed a porcine (pig) fibroblast cell line, in which the porcine homolog of Sall1 was knocked out using a targeting vector. The targeting construct has homology arms to sequences that flank exon 2 of Sall1 (Note: the homology arms are sufficient for targeting), and contains a modified exon 2 in which a neomycin resistance cassette (PGK-neo) has replaced a portion of exon 2 of the Sall1 gene. The relative positions of several PCR primers (A-F) are indicated. The targeting construct (an 8.6 kb Notl - Sall fragment) was transformed into porcine ear fibroblast cells and 515 neomycin resistant colonies were recovered. Sal-like 1 locus (14.4kb) H E1 Knock-out vector Targeted locus F 5.5kb PCR primer C BamHI PGK-neo PCR primer E PCR primer F FGK-neo PCR primerD PCR primer A PCR primer B (a) The neomycin resistant colonies were screened by PCR using the primers A and B. Only seven of the 515 colonies (AB1-AB7) resulted in the production of a PCR product of the expected size. The remaining 508 colonies failed to produce a PCR product (of any size). Explain why this would have been observed. Note: there is a rational explanation for this other than "the experiment did not work or there was a problem with the reagents". (b) Suggest how the researchers could have altered their targeting construct, to ensure that all of the neomycin resistant transformants would have generated the expected PCR product with the A/B primer pair.
The Sal-like 1 gene (Sall1) plays a role in kidney development. Point mutations in the gene result in abnormalities in the kidney and mice lacking the Sall1 gene show agenesis (absence) of the kidneys. As a preliminary step towards the generation of a human kidney in a pig, researchers developed a porcine (pig) fibroblast cell line, in which the porcine homolog of Sall1 was knocked out using a targeting vector. The targeting construct has homology arms to sequences that flank exon 2 of Sall1 (Note: the homology arms are sufficient for targeting), and contains a modified exon 2 in which a neomycin resistance cassette (PGK-neo) has replaced a portion of exon 2 of the Sall1 gene. The relative positions of several PCR primers (A-F) are indicated. The targeting construct (an 8.6 kb Notl - Sall fragment) was transformed into porcine ear fibroblast cells and 515 neomycin resistant colonies were recovered. Sal-like 1 locus (14.4kb) H E1 Knock-out vector Targeted locus F 5.5kb PCR primer C BamHI PGK-neo PCR primer E PCR primer F FGK-neo PCR primerD PCR primer A PCR primer B (a) The neomycin resistant colonies were screened by PCR using the primers A and B. Only seven of the 515 colonies (AB1-AB7) resulted in the production of a PCR product of the expected size. The remaining 508 colonies failed to produce a PCR product (of any size). Explain why this would have been observed. Note: there is a rational explanation for this other than "the experiment did not work or there was a problem with the reagents". (b) Suggest how the researchers could have altered their targeting construct, to ensure that all of the neomycin resistant transformants would have generated the expected PCR product with the A/B primer pair.
Human Physiology: From Cells to Systems (MindTap Course List)
9th Edition
ISBN:9781285866932
Author:Lauralee Sherwood
Publisher:Lauralee Sherwood
Chapter11: The Blood
Section: Chapter Questions
Problem 7UC
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