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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.

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 Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
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,...
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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.
Irreversible organ failure remains a worldwide concern as demand for transplantable organs far outpaces the available supply. To overcome this shortage, xenotransplantation, where tissues or organs from animals are transplanted into humans has been tested with limited success since the early 1900's. Factors affecting the outcome of transplantation across the xenogeneic barrier include both humoral (innate) and cell-mediated (innate/adaptive) immune responses. Despite the development of strategies to achieve immunological tolerance, xenotransplantation is not yet a clinical reality. Indeed, while organ rejection can be avoided, the risk of an infectious or zoonotic disease spreading from animals to humans raises further concerns about the clinical application of xenotransplantation. By the late 1990s, the advent of stem cell research had sparked a new direction in the field of regenerative medicine. As an alternative to generating organs from induced pluripotent stem cells (iPSCS) in vitro, providing iPSCs with an empty developmental niche for the specific organ might be beneficial. Indeed, according to the organ niche theory, developmental failure in organogenesis caused by defective genes, can be compensated by xenogeneic pluripotent stem cell transplantation into genetically affected blastocysts. First described in 1993, this blastocyst complementation technique generates an organ almost entirely composed of cells derived from the donor pluripotent stem cells. So, it may be possible then, to generate an entirely human kidney for example, in a pig that lacks the ability to generate a porcine kidney. A key step in this process would be the development of the so called "empty organ niche". 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) E1 Knock-out vector Targeted locus 5.5kb PCR primer C BamHI 1.3kb PGK-neo PCR primer E PCR primer F 1.8kb Sall PGK-neo E3 PCR primerD PCR primer A PCR primer B
Transcribed Image Text:Irreversible organ failure remains a worldwide concern as demand for transplantable organs far outpaces the available supply. To overcome this shortage, xenotransplantation, where tissues or organs from animals are transplanted into humans has been tested with limited success since the early 1900's. Factors affecting the outcome of transplantation across the xenogeneic barrier include both humoral (innate) and cell-mediated (innate/adaptive) immune responses. Despite the development of strategies to achieve immunological tolerance, xenotransplantation is not yet a clinical reality. Indeed, while organ rejection can be avoided, the risk of an infectious or zoonotic disease spreading from animals to humans raises further concerns about the clinical application of xenotransplantation. By the late 1990s, the advent of stem cell research had sparked a new direction in the field of regenerative medicine. As an alternative to generating organs from induced pluripotent stem cells (iPSCS) in vitro, providing iPSCs with an empty developmental niche for the specific organ might be beneficial. Indeed, according to the organ niche theory, developmental failure in organogenesis caused by defective genes, can be compensated by xenogeneic pluripotent stem cell transplantation into genetically affected blastocysts. First described in 1993, this blastocyst complementation technique generates an organ almost entirely composed of cells derived from the donor pluripotent stem cells. So, it may be possible then, to generate an entirely human kidney for example, in a pig that lacks the ability to generate a porcine kidney. A key step in this process would be the development of the so called "empty organ niche". 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) E1 Knock-out vector Targeted locus 5.5kb PCR primer C BamHI 1.3kb PGK-neo PCR primer E PCR primer F 1.8kb Sall PGK-neo E3 PCR primerD PCR primer A PCR primer B
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