SPEED技术介绍
丁香园论坛
1857
这个主意真不错，制备一个能表达siRNA的文库，无需知道靶mRNA 的序列就可以筛选出有效的siRNAs，可以按照不同的要求筛选出许多有用的序列，从而节约了大笔money.采用病毒为载体，可以象PHAGE表面展示技术一样易于筛选。
Small interfering RNAs (siRNAs) potently silence expression of
target genes. In principle siRNA libraries can be used to perform
effective genome-scale functional genetic screens in mammalian
cells, but their development has been hampered by the need to
chemically synthesize thousands of oligonucleotides and to incorporate
them into expression vectors. We have developed a technology
to efficiently convert a double-stranded cDNA library into
a retroviral siRNA library in which 21-base siRNAs are produced in
infected cells at high levels and efficiently block expression of their
target genes. The key steps are the generation of random cDNA
fragments that are fused to a hairpin linker, cleavage with the
MmeI endonuclease that creates 20- to 21-bp cDNA fragments,
conversion to a double-stranded DNA that contains two copies of
the cDNA insert in a head-to-head palindrome, and insertion of the
construct downstream of a polymerase III promoter. We constructed
a siRNA library with 3 106 clones from a mouse embryo
cDNA library; siRNAs were found against many different genes;
and multiple siRNAs can be generated from a single mRNA. We
further showed that specific siRNAs were efficiently produced in
stably infected mammalian cells and resulted in significant and
specific reduction of their target mRNAs. Because no prior knowledge
about target transcripts is needed, a cDNA-derived siRNA
library will generate siRNAs against unknown transcripts and
genes. Finally, cDNA-derived siRNA libraries can be readily generated
from any cell type or species, enabling genome-wide functional
screens in many biological systems.
5494–5499 PNAS April 13, 2004 vol. 101 no. 15
Small interfering RNAs (siRNAs) potently silence expression of
target genes. In principle siRNA libraries can be used to perform
effective genome-scale functional genetic screens in mammalian
cells, but their development has been hampered by the need to
chemically synthesize thousands of oligonucleotides and to incorporate
them into expression vectors. We have developed a technology
to efficiently convert a double-stranded cDNA library into
a retroviral siRNA library in which 21-base siRNAs are produced in
infected cells at high levels and efficiently block expression of their
target genes. The key steps are the generation of random cDNA
fragments that are fused to a hairpin linker, cleavage with the
MmeI endonuclease that creates 20- to 21-bp cDNA fragments,
conversion to a double-stranded DNA that contains two copies of
the cDNA insert in a head-to-head palindrome, and insertion of the
construct downstream of a polymerase III promoter. We constructed
a siRNA library with 3 106 clones from a mouse embryo
cDNA library; siRNAs were found against many different genes;
and multiple siRNAs can be generated from a single mRNA. We
further showed that specific siRNAs were efficiently produced in
stably infected mammalian cells and resulted in significant and
specific reduction of their target mRNAs. Because no prior knowledge
about target transcripts is needed, a cDNA-derived siRNA
library will generate siRNAs against unknown transcripts and
genes. Finally, cDNA-derived siRNA libraries can be readily generated
from any cell type or species, enabling genome-wide functional
screens in many biological systems.
5494–5499 PNAS April 13, 2004 vol. 101 no. 15
