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-like 1 locus (14.4kb)HE1Knock-out vectorTargeted locus5.5kbPCR primer CBamHI1.3kbPGK-neoPCR primer E PCR primer FSmal1.8kbE3PGK-neoPCR primerD PCR primer APCR primer B (c) Genomic DNA from one of the seven neomycin resistant clones, AB1, that produced a PCR productwith primers A/B was further examined by PCR alongside genomic DNA from neomycin resistantfibroblasts that did not generate a PCR product with primers A and B and untransformed porcinefibroblast cells. Clearly and neatly diagram an image of the agarose gel indicating the PCRproduct(s) that would be generated from each of the three genomic DNA templates when PCR isperformed with primers C and D. Illustrate the fragments and their sizes that would have beenvisualized on the gel. If sizes cannot be determined, relative position on the gel of differentfragments is critical.(d) If one reconsiders the results obtained above in (c) using the primer pair C/D on genomic DNAisolated from clone AB1 and assumes the rare event where there has been a biallelic targetingevent, would the results be different than those reported in (c) for AB1? Demonstrate your answerby clearly and neatly drawing an image of the agarose gel indicating the PCR product(s) that wouldbe generated from an AB1 genomic DNA template when PCR is performed with primers C and D ifthere had been monoallelic targeting and biallelic targeting. Illustrate the fragments and their sizesthat would have been visualized on the gel. If sizes cannot be determined, relative position on thegel of different fragments is critical.

Neomycin:Neomycin is an antibiotic medication used to treat or prevent bacterial infections. It…

(c) Genomic DNA from one of the seven neomycin resistant clones, AB1, that produced a PCR product with primers A/B was further examined by PCR alongside genomic DNA from neomycin resistant fibroblasts that did not generate a PCR product with primers A and B and untransformed porcine fibroblast cells. Clearly and neatly diagram an image of the agarose gel indicating the PCR product(s) that would be generated from each of the three genomic DNA templates when PCR is performed with primers C and D. Illustrate the fragments and their sizes that would have been visualized on the gel. If sizes cannot be determined, relative position on the gel of different fragments is critical. (d) If one reconsiders the results obtained above in (c) using the primer pair C/D on genomic DNA isolated from clone AB1 and assumes the rare event where there has been a biallelic targeting event, would the results be different than those reported in (c) for AB1? Demonstrate your answer by clearly and neatly drawing an image of the agarose gel indicating the PCR product(s) that would be generated from an AB1 genomic DNA template when PCR is performed with primers C and D if there had been monoallelic targeting and biallelic targeting. Illustrate the fragments and their sizes that would have been visualized on the gel. If sizes cannot be determined, relative position on the gel of different fragments is critical.

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,...