co-amplified in the same reaction mixture as an internal PCR 
control. 
Cells expressing antisense RNA showed complete inhibition of 
K-ras mRNA synthesis. There was no change in H-ras or N-ras 
expression in either antisense or sense transfectants. Antisense 
transfectants showed a 3-fold reduction in growth rate 
compared to sense transfectants and parental H460a cells but 
continued to grow in culture. Expression of antisense K-ras 
RNA reduced the growth rate of H460a tumors in nu/nu mice. 
Tumorigenicity of cell lines expressing antisense RNA was 
assessed by subcutaneous injection of 10 5 cells in nu/nu mice. 
Unmodified H460a cells formed tumors in all mice in 15 days. 
No tumors developed in mice injected with H460a antisense 
cells during 120 days of observation, whereas H460a cells 
transfected with Apr-1 -neo sense plasmid formed tumors similar 
to those formed by unmodified H460a cells. These experiments 
show that in H460a cells engineered to synthesize antisense K- 
ras RNA, the levels of K-ras mRNA and K-ras p21 protein were 
dramatically reduced. Our studies show that a construct can be 
made that distinguishes among members of the ras family. 
Previous studies with ras AS oligonucleotides showed inhibition 
of total p21 expression which led to cell death 23 , whereas our 
data show that antisense RNA generated from the genomic DNA 
of the K-ras gene can specifically inhibit K-ras expression. 
Inhibition of K-ras reduced the growth rate of H460a cells but 
did not alter cell viability or continued growth in culture, which 
suggests that redundancy in p21 expression may compensate 
for absence of expression by one member of this family so that 
functions essential for maintenance of cell viability are preserved. 
When antisense K-ras was transfected into H322a cells, which 
are homozygous for wtK-ras, the cells were unchanged in 
viability or rate of growth. These studies provide evidence that 
reduction in the expression of a single mutant gene product can 
reduce tumor cell proliferation and tumorigenicity. 
2.3. 1.2 Gene construct 
The retroviral vector construct contains the AS-K -ras fragment 
with its /?-actin promoter inserted into the LNSX vector 24,25 . The 
orientation of the insert is such that the transcription of the AS- 
K-ras is driven by the /?-actin promoter downstream from the 
insert. For this protocol the LNSX vector will be used initially 
and subsequently, when availabe, the G1 vector will be used. 
The differences in the G1 vector and LNSX are minor and 
should not alter the efficacy of the retroviral construct. 
Recombinant DNA Research, Volume 16 
[423] 
