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”. 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)E1Knock-out vectorTargeted locus5.5kbPCR primer CBamHI1.3kbPGK-neoPCR primer E PCR primer F1.8kbSallPGK-neoE3PCR primerD PCR primer APCR primer B

Question

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”. 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 &#34;empty organ niche&#34;.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)E1Knock-out vectorTargeted locus5.5kbPCR primer CBamHI1.3kbPGK-neoPCR primer E PCR primer F1.8kbSallPGK-neoE3PCR primerD PCR primer APCR primer B

Accepted Answer

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