A Method for gene "targeting" in zebrafish

Michael Lardelli Michael.Lardelli at bmc.uu.se
Sat Apr 20 07:32:20 EST 1996

Hi out there!

I would like to suggest a strategy for gene "targeting" in zebrafish. I
realise that I am probably going out on a limb by doing this. The method
(which seems rather obvious to me) may not be feasible for some reason that
I have not realised or it may simply be that news travels so slowly up to
Sweden that I have not heard of several other people who have
suggested/already begun identical or similar experiments. If it cannot be
done, please tell me what's wrong with it!  Anyway, here goes......

The idea is not really for a method of targeted mutation - rather, for a
kind of "targeted selection" for mutations in genes that cannot easily be
isolated by, for example, phenotypic selection. The reasoning goes like
this - as shown by Mullins et al. (Current Biology 1994, 4:189-202) and
Solnica-Krezel et al. (Genetics 1994, 136: 1401-20) very high rates of
mutation in genes controlling pigment synthesis can be induced in
premeiotic germ cells using the mutagen N-ethyl-N-nitrosurea (ENU). Indeed,
rates of one new allele per 1000 haploid genomes screened or higher have
been reported. Personally, I find this rate rather astonishing since one
would expect that most point mutations in a gene would be silent.

Anyway, the problem of mutating a zebrafish gene of choice may not be one
of mutagenesis but, rather, of selection. Without a phenotypic test the
mutation is invisible.

Why not mutate genomes with ENU and then look for the mutations directly at
the sequence level? If the frequency of null/hypomorphic mutations is so
high, or even if it were lower for particular genes (e.g. 1 in 10,000
haploid genomes screened) then it may still be reasonable to do a sequence
search. The problem, of course, is the technology.

Isogenic strains of zebrafish are available or it is easy to create fish
that are isogenic at a particular locus by pair-mating and inbreeding (if
isogenic strains are too "weak" to undergo efficient mutagenesis). The
steps in a "targeted selection" of the desired mutation in these fish might
be as follows:

1) Make a genomic library from the isogenic zebrafish strain and isolate a
'phage clone of the gene to be mutated.

2) Map the exons of the gene to be mutated. Design PCR tests that will only
amplify up exonic sequences. Ideally, the PCR tests could produce fragments
of less than 1000 bp (see below). Multiple PCR tests to amplify up multiple
exons could be designed.

3) Expose male isogenic fish to ENU to mutate premeiotic germ cells, wait
for sperm to be produced from these cells, and then mate with isogenic
female fish.

4) Collect the fry (that will be heterozygous for any induced mutations).
Pool them in groups of say, 10 fish each. At this stage (depending upon
system capacity and efficiency of DNA isolation) DNA can be taken from the
fry by the dipping method from Hopkin's lab or the fry can be grown up for

5) PCR Amplify the exons from the pools of fish (or from individual fish if
necessary) and screen for single bp changes using, for example, the
recently advertised "Mismatch Detect II kit" from Ambion that, (so they
claim), allows rapid screening of 0.5-1kb regions of DNA for single bp
changes (Their method relies on transcribing both a control and test DNA
fragment, hybridising the RNAs and then digesting with an RNAse that will
cleave the dsRNA at mismatches. The products are then analysed by
electrophoresis through agarose). I would envisage that hundreds of pools
of fish could be analysed in one week (or one day?) by this method. Thus,
within a matter of weeks one could examine tens of thousands of genomes for
single bp changes.

6) When a pool of fish is found to contain a mutation (i.e. is "positive")
the pool is divided up and retested to find the positive fish.

7) The relevant exon is amplified from the positive fish and then
sequenced. The mutation should be easy to identify (especially with
automated fluorescent dye sequencing) even though it will be present in a
"heterozygous" state in the PCR product.

8) If the mutation is found to be interesting (i.e. causes termination of
the protein or an interesting change in the protein sequence) then a PCR
test is designed that specifically identifies the mutated sequence. The
fish is then outcrossed for a couple of generations to remove mutations in
other genes (while the desired mutation is followed using the PCR test)
before inbreeding the desired mutation to homozygosity to observe its

There are a number of things to note with this strategy:

A) The technology is simple.

B) The suggested strategy should be rather rapid. I imagine that one could
go from mutagenesis to heterozygous mutant in about one year, i.e. about as
quickly as you can knock out a gene in mice (if you are fast).

C) Depending upon the isogenicity of the fish, simultaneous screening for
mutations in a number of genes is possible. Indeed, imagine the following
scenario - 100,000 heterozygous mutant male fish (representing 100,000
mutant haploid genomes) are generated as described above. Each fish is
given a number, sperm is taken and a "sperm bank" is assembled. Eventually,
each fish is used to prepare a large amount of genomic DNA that is placed
in a corresponding "DNA bank". This sperm/DNA bank might contain between
10-100 (or more) null/hypomorphic mutations for any gene. Then, whenever
you wish to find a mutation in a particular gene, you simply test the DNA
from the bank using the above method. When you find a suitable mutation the
sperm is taken and.....

Well, what do you all think? I would very much appreciate your comments on
this idea.

Yours sincerely,

Michael Lardelli

Michael Lardelli

Department of Developmental Neuroscience
Box 587
BioMedical Center
Uppsala University
S-751 23  UPPSALA

Tel.    (+46 18) 17 4133  -office
        (+46 18) 463286   -home
        Answering machines are coupled
        to both numbers.

FAX.    (+46 18) 559017

email:  Michael.Lardelli at bmc.uu.se

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