Irreversible organ failure remains a worldwide concern as demand for transplantable organs far outpaces theavailable supply. To overcome this shortage, xenotransplantation, where tissues or organs from animals aretransplanted into humans has been tested with limited success since the early 1900's. Factors affecting theoutcome of transplantation across the xenogeneic barrier include both humoral (innate) and cell-mediated(innate/adaptive) immune responses. Despite the development of strategies to achieve immunologicaltolerance, xenotransplantation is not yet a clinical reality. Indeed, while organ rejection can be avoided, therisk of an infectious or zoonotic disease spreading from animals to humans raises further concerns about theclinical application of xenotransplantation. By the late 1990s, the advent of stem cell research had sparked anew direction in the field of regenerative medicine. As an alternative to generating organs from inducedpluripotent stem cells (iPSCS) in vitro, providing iPSCs with an empty developmental niche for the specificorgan might be beneficial. Indeed, according to the organ niche theory, developmental failure inorganogenesis caused by defective genes, can be compensated by xenogeneic pluripotent stem celltransplantation into genetically affected blastocysts. First described in 1993, this blastocystcomplementation technique generates an organ almost entirely composed of cells derived from the donorpluripotent stem cells. So, it may be possible then, to generate an entirely human kidney for example, in a pigthat lacks the ability to generate a porcine kidney. A key step in this process would be the development of theso called "empty organ niche".The Sal-like 1 gene (Sall1) plays a role in kidney development. Point mutations in the gene result inabnormalities in the kidney and mice lacking the Sall1 gene show agenesis (absence) of the kidneys. As apreliminary 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 armsare 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) areindicated. The targeting construct (an 8.6 kb Notl - Sall fragment) was transformed into porcine ear fibroblastcells and 515 neomycin resistant colonies were recovered.Sal-ike 1 locus (14.4kb)E1Knock-out vectorTargeted locus5.5kbPCR primer CPGK-neoPCR primer E PCR primer FPGK-neoPCR primerD PCR primer APCR primer B(a) The neomycin resistant colonies were screened by PCR using the primers A and B. Only seven ofthe 515 colonies (AB1-AB7) resulted in the production of a PCR product of the expected size. Theremaining 508 colonies failed to produce a PCR product (of any size). Explain why this would havebeen observed. Note: there is a rational explanation for this other than "the experiment did notwork or there was a problem with the reagents".(b) Suggest how the researchers could have altered their targeting construct, to ensure that all of theneomycin resistant transformants would have generated the expected PCR product with the A/Bprimer pair..

In genetic engineering experiments, the antibiotic neomycin is frequently utilized as a selectable…

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.

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...