#gene targeting

Gene Tar getting

Which element is not necessary for gene targeting via homologous recombination?

Homologous recombination guided gene targeting requires extensive homology arms matching endogenous locus to direct strand exchange, and selectable marker like neo to identify integration events. Endogenous recombination machinery of embryonic stem cells including Rad51 mediated repair executes crossover. Origin of replication is plasmid bacterial element needed for propagation in E coli during vector construction and preparation but dispensable inside mammalian genome since integration occurs via chromosomal recombination, not autonomous replication. Therefore Ori is not required for actual gene targeting event in eukaryotic nucleus.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

What happens if the targeting vector lacks neor but contains HSV-tk?

Neomycin resistance gene neo confers survival in G418 or Geneticin through aminoglycoside phosphotransferase inactivating the drug. HSV-tk alone cannot provide G418 protection; it only sensitizes cells to ganciclovir via toxic metabolite formation. A vector lacking neo but retaining tk outside homology regions possesses no positive selection marker, so electroporated embryonic stem cells cannot detoxify G418 effectively. Even if random integration inserts tk, cells remain sensitive to G418 and die during selection. Therefore loss of positive marker aborts enrichment of targeted clones altogether preventing isolation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

In positive-negative selection, only cells with homologous recombination will:

Gene targeting vectors carry neo inside homology arms and HSV-tk outside. Any random integration retains both cassettes, making cells G418 resistant but ganciclovir sensitive because tk converts ganciclovir to a cytotoxic form. Homologous recombination replaces the genomic locus through double crossover, incorporating neo but losing tk sequences. Consequently only homologous recombinants acquire G418 resistance due to neo expression while avoiding tk-mediated toxicity. Growth in medium containing both G418 and ganciclovir therefore selectively enriches the rare correctly targeted embryonic stem cell clones.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

Which gene serves as a negative selection marker in gene targeting experiments?

Positive-negative selection enriches correctly targeted embryonic stem cells for knockout mouse production. The neomycin phosphotransferase gene placed inside homology arms confers G418 resistance, marking any vector integration event. Herpes simplex virus thymidine kinase located outside homology arms provides negative selection. Random integrants retain tk, which phosphorylates ganciclovir into toxic triphosphate nucleotides triggering cell death. Homologous double crossover excludes tk sequences, so true recombinants survive ganciclovir treatment while remaining G418 resistant, eliminating majority of non-specific insertions and ensuring isolation of correctly targeted clones.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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