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    10MCBI2021

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    of 21

    Reverse Genetics and

    Gene Knockouts

    This meeting is being


    recorded.

    Molecular and Cell Biology


    I BIOL-UA 21
    Meeting 10
    12 October 2021
    Connecting genes and functions

    “classical” or
    “forward” genetics
    Figure 6.1
    Molecular Cell Biology, Eighth Edition
    “reverse” genetics
    © 2016 W.H. Freeman and Company

    2
    Knocking out a gene by homologous recombination

    Figure 6.37b
    Molecular Cell Biology, Ninth Edition
    © 2021 W.H. Freeman and Company 3
    2:0 segregation in progeny of a heterozygous diploid

    4
    What
    What isiswrong
    wrongwith
    withthe mutants? (cell morphology?)
    the
    mutants?

    Levy & Siegal, PLOS Biology 6:e264 5


    What is wrong with the mutants? (growth rate?)

    Matiza Sacotingo & Diego Quintana 6


    Embryonic stem cells can be genetically engineered

    Figure 22-4a
    Molecular Cell Biology, Ninth Edition
    © 2021 W.H. Freeman and Company 7
    Gene targeting in ES cells

    Figure 6.37a
    Molecular Cell Biology, Eighth Edition
    © 2016 W.H. Freeman and Company 8
    Making a knockout mouse: inject ES cells into embryo

    Figure 6.38
    Molecular Cell Biology, Eighth Edition
    © 2016 W.H. Freeman and Company 9
    1
    0
    Injecting ES cells into an embryo

    1
    Practice question 1

    You are knocking out the yeast LYS2 gene, which is


    required for production of the amino acid lysine. Yeast
    cells without the LYS2 gene will not grow unless their
    medium is supplemented with lysine.
    1) Draw a schematic diagram of the knockout construct you

    would use to knock out LYS2 and replace it with the


    selectable marker kanMX, which confers resistance to
    G418.
    2) Describe the procedure for knocking out LYS2 with this

    construct and selecting mutant cells.


    3) Describe how you could validate that the selected cells

    indeed contain the desired knockout.

    1
    Practice question 2

    1
    Need for conditional disruption of gene
    function What if a gene is required for early
    embryogenesis and for ear development?

    1
    Two common methods of conditional disruption

    knockout a gene during


    development so it is
    missing only in some
    tissues

    interfere with mRNA or its


    translation only in some
    tissues

    1
    Cre recombinase catalyzes site-specific recombination

    white +
    no Cre present

    Cre present (in some cells)


    white +

    (lost during
    cell division)
    16
    Making a conditional knockout mouse with Cre/loxP

    Figure 6.39
    Molecular Cell Biology, Eighth Edition
    © 2016 W.H. Freeman and Company 1
    Highlight: BRCA1 conditional knockout
    mouse
    BRCA1 knockout is recessive embryonic lethal
    mammary-specific
    BRCA1 promoter

    What does this result say about tumor formation?


    What would the consequence be of replacing the
    mammary- specific promoter with a skin-specific
    promoter?
    What would the consequence be of replacing the
    mammary- specific promoter with a promoter that is only
    expressed in all cells of very early embryos?

    Trends in Genetics 17:S18 1


    Lowlight: Transgenic humans

    Second International Summit on Human Genome Editing (November 2018) 1

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