The high titer of virus generated from N2 and the presence of a cryptic 3* 
splice site necessary for the expression of the Neo gene is not unique to this 
vector construct. Three similar vectors, N1, N2 and N3 , all of which contain 
varying amounts of gag coding sequences preceding the Neo gene , generate higher 
titers of virus as compared to N4 or DE vectors. All three vectors activate a 
cryptic 3' splice site enabling the efficient expression of the Neo gene, 
although in each vector, a different 3' splice site is activated (unpublished 
results). It is therefore tempting to speculate that this region of the M-MuLV 
intron contains currently unidentified functions which may be important for the 
efficient replication of this virus. For example, sequences involved in 
packaging may extend into this region. 
N2 derived ADA vectors 
The usefulness of the N2 vector system for transfer and expression of 
nonselectable genes was tested next. In these studies, the human ADA cDNA was 
fused to a DNA fragment encoding the mouse metallothionein I (MT) promoter or 
the SV40 early promoter and was inserted into the N2 vector, downstream from the 
Neo gene, in the same transcriptional orientation as that of the LTR (Fig. IB, 
MAX and SAX, respectively). The titer of virus generated from the MAX and SAX 
vector was about 0.5-2.x10^ cfu/ml. Thus, the insertion of the MT-ADA or 
SV40-ADA DNA fragments in N2 had little, if any, effect on the otherwise high 
titer of the corresponding virus. 
Both MAX and SAX infected cells express the two LTR driven unspliced and 
spliced RNA forms (Fig. 2, panels B,C,D). Note that the insertion of the MT-ADA 
or the SV40-ADA into N2 (about 1400 bp downstream from the cryptic 3' splice 
site) results in a 5-10 fold reduction in the level of the aberrantly spliced 
RNA form, the presumptive mRNA for the Neo gene product (Fig. 2). This reduction 
is also reflected at the level of the Neo qene product (Fiq.3A) further 
Recombinant DNA Research, Volume 12 [255] 
