11. The following cassette was designed to create estrogen receptor knock-out mice:
SoH: site of homology; Gol: gene of interest
What would ensure that proper recombination has taken place?
(1) Cells survive when cultured in presence of only G418
(2) Cells survive when cultured in presence of G418 followed by ganciclovir
(3) Cells die when cultured in presence of G418
(4) Cells survive when cultured with G418 and die when cultured with ganciclovir
The correct answer is option (2): cells that have undergone proper homologous recombination will survive in the presence of G418 and will also survive after subsequent culture in ganciclovir.
Question recap and concept
The cassette contains, between two sites of homology (SoH), a disrupted estrogen receptor gene of interest (GoI) linked to a neomycin‑resistance gene (neo⁺), while a herpes simplex virus thymidine kinase gene (tk) lies outside the homology region. In ES‑cell gene targeting, neo⁺ provides positive selection with G418, whereas tk provides negative selection with ganciclovir because tk converts ganciclovir into a toxic nucleotide that kills cells expressing tk.
Homologous recombination at the estrogen receptor locus integrates only the segment between the homology arms, so neo⁺ is inserted into the target gene but tk is left behind on the non‑integrated vector; random integration typically inserts both neo⁺ and tk together.
Option (1): Only G418 survival
Statement: “Cells survive when cultured in presence of only G418.”
G418 kills cells unless they express the neomycin‑resistance gene (neo⁺), so both correctly targeted clones and randomly integrated clones will be G418‑resistant. Because G418 selection alone cannot distinguish homologous recombinants (neo⁺, tk⁻) from random integrants (neo⁺, tk⁺), survival only in G418 does not ensure proper recombination.
Therefore, option (1) is insufficient and thus incorrect.
Option (2): G418 then ganciclovir survival (Correct)
Statement: “Cells survive when cultured in presence of G418 followed by ganciclovir.”
After transfection, initial culture in G418 selects all cells that have stably acquired neo⁺, including both homologous recombinants and random integrants. Subsequent treatment with ganciclovir kills cells that express tk, so random integrants (neo⁺, tk⁺) die, whereas correctly targeted clones (neo⁺, tk⁻) survive because tk was never inserted into their genome.
Thus, survival after both positive (G418) and negative (ganciclovir) selection specifically enriches for correctly targeted ES cells, making option (2) the correct answer.
Option (3): Death in G418
Statement: “Cells die when cultured in presence of G418.”
Cells that have incorporated the targeting construct by either homologous or random integration will express neo⁺ and should therefore survive G418; death in G418 simply indicates that no functional neo⁺ gene is present. Such cells cannot represent successful gene targeting, so this outcome does not indicate proper recombination and makes option (3) clearly wrong.
Option (4): Survive G418 but die in ganciclovir
Statement: “Cells survive when cultured with G418 and die when cultured with ganciclovir.”
Clones that are G418‑resistant but ganciclovir‑sensitive must express both neo⁺ and tk, which is the hallmark of random integration where the entire cassette, including tk outside the homology arms, is inserted at ectopic sites. Such cells have not undergone precise homologous recombination at the estrogen receptor locus, so option (4) actually selects against the desired targeted clones and is incorrect.
SEO‑friendly introduction
Gene targeting in embryonic stem cells for creating estrogen receptor knockout mice commonly uses a cassette carrying neo⁺ within the gene of interest and tk outside the homology arms to enable positive‑negative selection. Correctly designed selection with G418 and ganciclovir enriches for ES‑cell clones that have undergone precise homologous recombination at the estrogen receptor locus while eliminating random integrants.
