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Fig. 3. Targeted deletion of mD2LIC. (A) The targeting strategy involved
removing 2.75 kb of the endogenous mD2LIC locus. This contains 500 bp
upstream of the predicted transcriptional start site together with the first
two exons, which include the translation start site and half the P-loop
domain. The targeting vector incorporates both positive (neomycin resistance)
and negative (diphtheria toxin A) selection, and comprises a 5.5 kb 5'
homology arm, a 2 kb loxP flanked PGKneo cassette (which
replaces the targeted region) and a 2.3 kb 3' homology arm. During the
targeting event, an exogenous HindIII restriction site is introduced
into the locus just downstream of the PGKneo cassette. (B) Use of a
3' probe in Southern blot analyses of transfected ES cell genomic DNA
digested with HindIII reveals a 13 kb band corresponding to the
wild-type locus and a 6 kb band representing a correctly targeted locus. (C)
Northern blot analysis of littermates from heterozygous crosses demonstrates
loss of the mD2LIC transcript in homozygous mutant individuals and a
decrease in heterozygous animals. (D) Primers P1 and P2 (indicated in A),
together with a pair of primers designed to detect neo, distinguish
between targeted and non-targeted alleles when genotyping. (E) Classification
of mD2LIC–/– mutants into three groups
according to the extent of embryonic turning at 9.5 dpc
(Table 1). Class I mutants
(bottom, 61%; n=39), which exhibit the most severe phenotype, fail to
initiate embryonic turning. Three examples are shown here. Class II (middle,
21%) mutants start but do not complete embryonic turning, and Class III (top,
18%) mutants complete turning but always display an open neural tube in the
region of the head (arrowhead). Other defects include reversal of heart
looping (Class II, white arrowhead), ballooning of the pericardial sac,
anterior truncations (Class III, white arrowhead) and defects in truck and
tail development.
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