Table 2. Single-cell RT–PCR results cDNA type Ly49DB6 Ly49DNOD Ly49DB6 and NOD Number of transcribing 5 13 22 single cells (n) Percentage positive 5/40=12.5% 13/40=32.5% 22/40=55% 40/73=55% 33/73=45% 7 7/80=8.8% Nucleic Acids Research, 2009, Vol. 37, No. 16 5337 Total Ly49D Ly49D+ NKG2D+ 40 40 Ly49D+ NKG2D+ 33 Total Ly49D+ NKG2D+ NKG2D 73 Total cells with cDNA 80 contained carrier cells. The single-cell RT–PCR technique will underestimate bi-allelic expression due to the fact that one allele may not be detected through ineﬃcient cDNA generation during the RT reaction, multiple sample puri- ﬁcation steps and random sampling error (27,39). As a control for the eﬃciency of our method, we used Nkg2d, an activating receptor gene that is transcribed in all NK cells (40) most probably at levels similar to the Ly49 genes, to gauge the eﬃciency of cell sorting, cDNA generation and detection. We only detected 73 Nkg2d+ cells from the total cells assayed after multiple sets of inde- pendent PCRs (Table 2). Nkg2d transcripts went unde- tected in seven NK cells indicating that possibly 9% of the time the transcript of a gene and/or allele is not detected by our method. In total, we detected transcript for at least one of Ly49dB6, Ly49dNOD and Nkg2d in 80 cells from the total of 88 NK cells assayed. Interestingly, the diﬀerence between the fraction of cells positive for Ly49dNOD versus Ly49dB6 is nearly 3-fold. The allelic expression of Ly49G in various F1 hybrid mice using allele-speciﬁc antibodies shows that diﬀerent alleles of the same gene can be expressed at diﬀerent levels in various mouse strains (41). This might be due to the MHC class-I background of the mice, the Ly49 cluster gene composition as well as polymorphisms in the pro- moter regions. Expression of Ly49H in a single genomic allele mouse model Ly49H is expressed on 50% of splenic NK cells in the B6 strain. For a completely bi-allelically transcribed gene expressed in 50% of NK cells, each allele would be expressed in all expressing cells and would contribute half of the total transcript in each cell. In a situation with only one genomic allele present, the fraction of cells expressing the gene should, however, remain the same at 50% but the amount of total transcript should be halved. For a gene expressed mono-allelically through allelic exclusion, one allele is chosen for expression and the other allele is repressed. If this gene is expressed in 50% of cells, each allele is transcribed in 25% of cells only. In contrast, for a stochastic mono-allelic expression pat- tern, as described for the inhibitory Ly49 receptors, every allele is equally, randomly and independently expressed (42). Based on this model, if 50% of NK cells are positive for a receptor, each allele should be expressed in 29.2% of NK cells with 8.4% of all NK cells expressing this receptor bi-allelically (see model of this situation in Figure 5A). We attempted to distinguish between the bi-allelic versus stochastic mono-allelic model using the B6.BXD8 (B6.Ly49h / ) mouse, generated recently by two indepen- dent groups (23,43), which carries the B6 Ly49 cluster with a 26 kb deletion spanning the length of the Ly49h gene. Both groups have independently shown that the expres- sion of other Ly49 receptors does not change in the B6.BXD8 mouse compared to the wild-type B6 mouse indicating that the lack of Ly49H does not aﬀect the expression of other Ly49 receptors (23,43). Unfortunately, an inhibitory Ly49 gene deletion mouse model does not exist for comparison. The B6.BXD8 is unique because the deleted Ly49 genomic allele is in the same Ly49 gene cluster as the other (non-deleted) chromosome. The vari- able Ly49 clusters as well as possible diﬀerences in MHC class-I background can produce confounding eﬀects on expression of Ly49 receptors in F1 hybrids (41). To investigate the expression pattern of Ly49H on NK cells carrying only one genomic allele of Ly49h on the B6 background, we FACS analyzed peripheral blood NK cells from 6-week old Ly49 h+/ heterozygous (B6.BXD8/B6) mice by staining with the 3D10 antibody. The percentage of NK cells expressing Ly49H in the het- erozygous mice (Ly49h+/ ) was reduced to 35% from that of age-matched B6 (Ly49h+/+) that have 50% Ly49H+ NK cells (P= 0.0005) (Figure 5B). This fraction of Ly49H expressing cells in the heterozygotes is lower than expected for a completely bi-allelically expressed gene but higher than the expected 29.2% based on the stochastic mono- allelic model (Figure 5A). Thus, based on these data, the situation for Ly49h likely lies between these two extremes. Although we cannot predict the possible eﬀects of the deletion of one Ly49h allele on the expression of the other allele, we can assume that in the wild-type B6 back- ground both alleles should have the same probability of expression. Since Ly49H is expressed on 50% of NK cells and assuming that in the Ly49h+/+ B6 mouse the second allele would also be expressed on 35% of NK cells, the bi-allelic expression of Ly49H would be 20% (Figure 5C). This value is strikingly similar to the percent- age of Ly49dB6/NOD (bi-allelic) NK cells (assuming 50% Ly49D+ NK) of the B6/NOD F1 hybrid, as assayed with single-cell RT–PCR (Figure 5D). Low sequence identity in the 5’-UTR of inhibitory and activating Ly49 genes The regulatory elements governing the transcription of activating Ly49 genes have not been analyzed to date. It is unknown if inhibitory and activating genes share common transcription factors. Multiple alignment of a number of inhibitory and activating Ly49 genes from var- ious sequenced mouse strains (performed with Clustal W)