affect facial or cardiac phenotypes concluding that
expression in neural crestcells is not essential for normal development.However, by placing this conditional knockout on a systemic null
backgroundthe embryos do in fact have both facial and exacerbated cardiac phenotypes. Thispoints to the idea that a gene dosage relationship is important in neural crestdifferentiation.
Placental Lactogen1 (
) is a critical growth factor with regard to both embryonicand extra-embryonic tissue. In
is reduced causing vascularformation within the yolk sac to cease soon after it begins.The initial idea was to create a
marker allele that would not behaploinsufficient for
. In order to show Hand1-expressing cells, a cDNA-knock-in approach with an IRES eGFP cassette was undertaken. The
homozygous cDNA knock-in mice are not viable and the lethality is not rescued bythe removal of the neomycin cassette.It was however found that these homozygous knock-in embryos exhibit a definiteextention of survival to between E10.5 and E12.5; different from
systemicknock-outs. Phenotypic outcomes include irregular development of the caudalregions, thinner and more dilated hearts. Furthermore, due to
’s role in extra
-embryonic tissue the tissue and yolk sacs displayed phenotypes. Using mRNAanalysis it was found that
cDNA knock-in alleles are hypomorphic with
expression being between 30% and 40% that of wildtype littermates. This isindicative of a threshold of necessary
expression. As was expected,
was upregulated when
expression decreased. This creates an imbalance withregard to bHLH stiochiometry.
Materials & Methods
targeting vector employed the
identical 5’ and 3’ targeting arms
employed in generation of the
knock-in (5). Inserted between these arms isa
beginning 70 base pairs 5’ of the
initiating methionine andending at the translational stop site. The
cDNA is immediately 5’ of an
IRESGFP cassette derived from pIRESeGFP
(Clonetech), which is immediately 5’ of
a PGKNeo cassette flanked by loxP sites. Homologous recombination was achieved ata frequency of one in fifteen and two independent ES lines were injected andproduced germline transmission with indistinguishable phenotypes.
Mouse strains Hand1Hand1
mice were generated by the IU knockout-transgenic core from EScells targeted with the construct described above.
mice weresubsequently generated via intercross of
mice with mice heterozygousfor