Worm Breeder's Gazette 14(1): 64 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Carnegie Institution of Washington, 115 West University Parkway, Baltimore Md. 21210.
Reporter gene constructs (e.g. lacZ and gfp fusions) have been
extremely useful in analyzing the regulation of gene expression in somatic
tissues of C. elegans. Somewhat surprisingly, however, gene fusions have
not been applicable (to date) for studies of expression in the germline.
The most definitive evidence for this "failure" of germline expression is
as follows: starting with a gene known to function in the germline, we
insert the gfp (or lacZ) coding region in frame without removing any
sequences from the original gene. When introduced by standard
transformation, these constructs showed no reporter activity in the
germline, although somatic activity is observed. Similar conclusions came
from large scale "gene-trap" screens: while many different somatic
patterns were recovered [1], no germ line expression has been reported in
any of these screens.
We suspect that several factors are combining to block germline
reporter expression. Assays for gene expression without a reporter
(genetic rescue assays) have been successful for some germline genes
(suggesting under some circumstances that tandem arrays can express in the
germline), but in other cases germline rescue has been weak or completely
unsuccessful. None of these genes have produced reporter fusions active
in the germline, suggesting that the reporter segment and the
transformation assay may each contribute to difficulty in germline
reporter expression.
In attempting to achieve more appropriate reporter expression, we
have been trying to produce transgenes which are as close as possible in
structure and context to endogenous chromosomal genes. We previously
reported modified lacZ and gfp vectors containing multiple intron
sequences [2]. These vectors yielded increased levels of somatic
activity, but did not solve the problem of germline expression.
Nonetheless, the intron-rich reporter segments seemed a good starting
point for additional exploration.
Several features make let-858 a good prototype gene to explore
modifications in transformation technique. LET-858 functions both in soma
and germline: transgene activity in soma is seen by rescue of embryonic
lethality, while germline function can be assayed by rescue of a
subsequent sterility defect (see previous abstract). LET-858 can be
tagged by inserting gfp in frame near the N-terminus.
When introduced into let-858 heterozygote strains by standard
transformation methods (co-transformed with rol-6d), we easily obtained
transgenic lines which expressed the let#030#858::gfp construct in all
somatic tissues. let-858 homozygote animals from these strains show
rescue of zygotic lethality, but show no rescue of the sterility defect.
No expression is observed in the germline of these sterile animals or
their healthy (let-858/+) siblings.
The standard transformation scheme results in the formation of
repetitive (head-to-tail) arrays with several hundred tandem interspersed
copies of the injected plasmids [3]. We reasoned that this structure
might be viewed by the animal as foreign or heterochromatic; we therefore
attempted to modify the transformation protocol to minimize the repetitive
nature of the transgene context. This was done by diluting the mixture of
rol-6 and let#030#858::gfp plasmids with C. elegans DNA. All three DNAs
were cleaved with restriction enzymes to promote formation of linear
"chromosomes" which should be complex in structure. We aimed at relative
concentrations expected to give approximately one molecule each of rol-6
and let-858::gfp per array. Injection of these mixtures produced very few
rolling animals in the first generation. Significant fractions of F1
transformed animals and of transgenic lines had specific morphological or
movement defects (presumably reflecting deleterious effects of the
randomly ligated N2 DNA segments). Nonetheless, we were able to obtain
transgenic lines [25-50% of the F1 rollers give rise to transformed
lines].
Approximately 50% of fluorescent let-858::gfp lines expressed in
both germline and soma. These had strong GFP activity throughout the
distal arm of the gonad, in oocytes, and in all stages of embryos. The
let-858 sterility defect was rescued in these lines.
To avoid the complexities of injecting C. elegans DNA, we've been
testing other sources of high-complexity DNA. DNA from the bacterium
Haemophilus influenza [4] was effective as carrier, allowing transgenic
lines to be obtained much more easily than with the use of C. elegans
carrier DNA. The transgenic lines produced with Haemophilus DNA have a
surprising property of progressive germline silencing: strong expression
can initially be seen in the germline, but after a few generations,
germline activity is greatly decreased. Similar losses of germline
expression may occur (albeit more slowly) in lines where C. elegans DNA
was used as carrier. The bacterial genome is less complex than C. elegans
but differs also in features such as repeat structure, GC content and
telomeres. We are trying a variety of other complex DNAs to 1) obtain a
universally applicable carrier and 2) test whether silencing of germline
activity is indeed correlated with array complexity.
The activity of the let-858::gfp construct gives a "foot-in-the-door"
to characterize, engineer and study germline expression. We're examining
sequence requirements for germline expression of the construct; this
should allow the production of vectors to express arbitrary coding regions
in germline. The ubiquitously expressed let-858 constructs may also serve
as a naive assay system for testing protein and RNA motifs from other
genes for their effect on activity level and localization pattern in the
germline and early embryos.
The complex arrays that are produced by co-transformation of diluted
plasmids with C. elegans genomic DNA appear also to provide improved
expression in somatic tissue. Preliminary observations suggest in several
cases that expression of transgenes (on a per-copy basis) is much stronger
than with traditional mixed arrays. We've also examined the ability of
the unc-54 promoter to enhance a myo-2 promoter which has been placed
adjacent. We had previously seen that this enhancement can be seen in
transient (F1) assays, but not in high copy number lines. The enhancement
is visible in complex arrays made by co-injection of C.elegans genomic
DNA. If generally applicable, this property should be very useful in
studies of somatic enhancer function.
[1] Hope, Development 113, 388; Young & Hope, Dev. Dynamics 196, 124;
Seydoux & Fire, wbg12#4, 20; Ishihara & Katsura C.elegans meeting
1993, p. 212.
[2] See Fire & Xu wbg 13#4, p. 20; Fire, Seydoux & Xu, C. elegans meeting
1995 p.213
[3] Stinchcomb, Shaw, Carr & Hirsh MCB 5, 3484; Mello, Kramer, Stinchcomb
& Ambros EmboJ 10, 3959.
[4] DNA from Haemophilus influenza was a kind gift of Hamilton Smith
(Johns Hopkins) The complete genome sequence of this strain has
recently been published, making this a reagent of known structure.
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