Wu et al. / J Zhejiang Univ SCI 2004 5(6):644-650
Targeting of human aFGF gene into silkworm,
Bombyx mori L. through homologous recombination*
WU Xiao-feng (吴小锋)†, CAO Cui-ping (曹翠平)
(Lab of Gene Engineering, College of Animal Sciences, Zhejiang University, Hangzhou 310029, China)
Received Oct. 29, 2003; revision accepted Jan. 14, 2004
Abstract: The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene
of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL1392
used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and
the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The
results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through ho-
Key words: Gene targeting, Recombinant vector, Bombyx mori L.
Document code: A CLC number: S881.2
Transgenic technology developed over the 20
years, has become a common tool widely applied to
livestock species because it is technically quite
simple, requiring only the injection of ‘naked’
DNA into the nucleus of a fertilized eggs (Hammer
et al., 1985; Suraokar and Bradley, 2000). However,
this technique has limitations in that the injected
DNA integrates randomly into the genome and thus
could not be expressed in the desired tissue or at the
appropriate level. More importantly, with this
technique, it is not possible to specifically modify
the genes of the species itself, although it can add
new genetic information. In order to overcome this
shortcoming, great effort was exerted to produce
gene-targeted animal in which the aim-gene was
inserted correctly into the desired locus on the
genome. Definitely, this approach has great sig-
nificance and might bring numerous biomedical
benefits: for example, ablation of xenoreactive
transplantation antigens, inactivation of genes re-
sponsible for neuropathogenic disease and precise
placement of transgenes designed to produce pro-
teins for human therapy. Gene-targeting was suc-
cessful firstly in mouse (Thompson et al., 1989) but
has not been achieved in other mammals because
functional embryonic stem (ES) cells like those in
mouse have not been derived until the latest report
(Suraokar and Bradley, 2000) of a breakthrough in
this field: gene-targeted sheep was successfully
produced by nuclear transfer from cultured somatic
cells which were treated by transfection and drug
selection in advance (McCreath et al., 2000).
Relatively, gene-targeting in insects lags be-
hind. Early studies indicated that the plasmid DNAs
injected into silkworm eggs were rapidly degraded
Journal of Zhejiang University SCIENCE
* Project supported by the Scientific Research Foundation for
Returned Overseas Chinese Scholars, Education Ministry of China
and the Natural Science Foundation of Zhejiang Province (No.
Wu et al. / J Zhejiang Univ SCI 2004 5(6):644-650
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the reporter gene GFP. DNA analyses of GFP-po-
sitive G1 silkworms revealed that multiple inde-
pendent insertions occurred frequently. The trans-
gene was stably transferred to the next generation
through normal Mendelian inheritance. This effi-
cient method of stable gene transfer in a lepidop-
teran insect opens the way for promising basic
research and biotechnological applications.
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