Having optimised the conditions for white cells enrichment, we decided to apply the Cell Sorter methodology to perform the screening (Fig 3,13). We calculated that a single enrichment by FACS would allow us to increase the number of clones tested per screen to 1.5x10^.
Briefly, twenty 10cm dishes were seeded with 5x10^ Melan-a cells and infected with the mouse cDNA library. Estimating an average infection efficiency o f -15% , this resulted in 75,000 cells infected per plate, meaning that 1.5x10^ clones were tested in
1) Infect Melan-a cells with mouse cDNA library 2) Hygromycin selection 3) White cells enrichment with FACS $ ♦ ♦ ♦ ♦ «
4) Wait two weeks for colonies to grow.
Visual screen of positive colonies (white colonies)
5) Subclone positive colonies and expand for genomic DNA/Provirus recovery
Positive colony
and the surviving cells were passed through the Cell Sorter gating for the
unpigmented ones. During the Hygromycin selection, the resistant cells divided ~5 times, so that by the time we were able to pass the cells trough the Cell Sorter, the total number of cells per plate was -2x10^. This forced us to sort each plate separately, the whole process taking -3 0 minutes per sample. The number of cells gated per sample was -15,000 (-0.8% gating), and these selected cells were plated at low density in six 10cm dishes for colony formation (2,500 cells per 10cm dish), making the total number of dishes 120. After 14 days, the dishes were visually
screened using a lightbox and 29 white and good-sized colonies (130 cells per colony) were subcloned. Although most of the clones remained unpigmented during the period of time necessary to expand them enough to allow genomic DNA extraction and pro virus recovery, only two (2al/5 and 8al/6) retested positively when re infected into melanocytes and assayed for melanin content (Fig 3.14).
Taken together these results suggest that the introduction of the enrichment step in our screening allowed the identification of positive clones by greatly enhancing the
number of clones tested. Moreover, the finding of genuine positive clones was a validation of the whole methodology.
100 n 90 80 - 70 - 60 ^ 2 Q. 50 Û) s 40 - 30 20 10 0 -ve Agouti 2a 1/6 8a 1/5
Fig 3.14 Relative melanin content in melanocytes infected with empty
3.6.1 Positive clones sequencing and identification
The cDNA of both positive clones was fully sequenced using the Dye terminator methodology. Sequence analysis revealed that the two positive clones were identical except for the length of the PolyA^ tail, indicating that they were different clones of the same transcript (Fig 3.15). This means that using our screening we were able to independently isolate the same gene twice, validating both the technology used and the ability o f the gene to cause the phenotype changes selected in our screening when overexpressed in melanocytes.
The gene sequence o f the two clones was compared to the available database of the NCBI Blast program. The result was a very good match with the published sequences
of the Em xl gene. The only mammalian Emxl sequences available in the database are
198bp of the mouse Emxl homeobox region (100% identity-Simeone et al, 1992),
301 bp o f the 5’ rat Em xl sequence (94% identity-Robel et al, 1995) and the last
462bp o f the human Emxl coding sequence (92% identity-Simeone et al, 1992) (Fig
- - I 10 2 0 30 40 50 6 0 7 0 8 0 90 100 2 « l / 6 j a a t t c g g c a c g a g g c c g g c c a c a g c c c g a t t a g c a a g g g a g a g a t c c a g g a g g a c c g c t g a a g c g g c c c t c a g t a g c a c a g g c a g c c c g c g c c c g g g c g 8 a l / 5 y a a t t e g g c a c g a g g c c g g c c a c a g c c c g a c c a g c a a g g g a g a g a c c c a g g a g g a c c g c c g a a g c g g c c c c c a g c a g c a c a g g c a g c c c g c g c c c g g g c g - I I I - I - I . - . . I - . . . I . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | , . . . | . . . , | . . . | . . . . | 110 120 130 140 1 5 0 160 1 7 0 1 8 0 1 9 0 2 0 0 2 a l / 6 c c a g a g a g g a g c c g c c c g c c g g g c c g c g g g g a c c a c g c c c c a g g c g g c g c g c a g c g g c c c a a c c c g g c g c c a g g g c c c c c g c a c g c c c c c c g c c g c c c c g 8 a l / 5 c c c g a g a g g a g c c g c c c g c c g g g c c g c g g g g a c c a c g c c c c a g g c g g c g c g c a g c g g c c c a a c c c g g c g c c a g g g c c c c c g c a c g c c c c c c g c c g c c c c g I - I . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . [ . . . . I 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 300 2 a l / 6 c c c c c g g c c g g c c c g c a a g g g c c g a g c g c g g g c g g g c g g g c g g g c g g g g g a g g c g a a g g g c g c g g g c g c g agcgcA TO TO CCAOCCTTOO TOCACACCCC 8 a l / 5 c c c c c g g c c g g c c c g c a a g g g c c g a g c g c g g g c g g g c g g g c g g g c g g g g g a g g c g a a g g g c g c g g g c g c g agcgcA TO TO CCAOOCTTOO TOCACACCCC
. . . . | . . . . | . . . , | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0 400 2 a l / 6 CCACOOCGOC AOCOCCGOOG CACCOAOCGT TCCCCAQAOC CCCGCTACCT CACTCCOCTT CGOCOOCCOC OACOATOTTC CAOCCCOCOO CTAAOCOOOO 8 a l / 5 OCACOOCOCC AOCOCCOOOG CACCOAOCGT TCCCCAQAOC CCCOCTACCT CACTCCOCTT CGOCOOCCOC OACOATOTTC CAOCCCOCOO CTAAOCOOOO
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . l . . . . | . . . . | . . . . | . . . . | . . . . 1 . . . . I . . . . | . . . . | . . . . | . . . . |
4 1 0 4 2 0 4 3 0 4 4 0 4 5 0 4 6 0 4 7 0 4 * 0 4 9 0 500 2 a l / 6 TTTCACCATA OAOTCCTTOO TOOCCAAOOA TOOTOOCACC OOCOGOAOTC CTOOCAOCOC OOOCOCOOOC TCCCATCCAC TOOCTOTOOC COCATCAOAO 8 a l / 5 TTTCACCATA OAOTCCTTOO TOOCCAAOOA TOOTOOCACC OOCOOOAOTC CTOOCAOCOC OOOCOCOOOC TCCCATCCAC TOOCTOTOOC COCATCAOAO
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . , | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
510 5 2 0 5 3 0 540 5 5 0 5 6 0 5 7 0 5 8 0 5 9 0 6 00 2 a l / 6 OAACCOCTCA OOCCCACOOC OCTCAACTAT CCTCACCCCA OTOCOOCCOA OACOOCCTTC OTOAOTOOCT TCCCTOCCOC TOCCOCTOCO OOCOCCOOCC 8 a l / 5 OAACCOCTCA OOCCCACOOC OCTCAACTAT CCTCACCCCA OTOCOOCCOA OACOOCCTTC OTOAOTOOCT TCCCTOCCOC TOCCOCTOCO OOCOCCOOCC
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . , | . . . . | . . . . | . . . . | . . . . | . . . . |
6 1 0 6 2 0 6 3 0 640 6 5 0 6 6 0 6 7 0 6 1 0 6 9 0 7 0 0 2 a l / 6 OCTCOCTCTA TOOAOOOCCO OAOCTOOTOT TCCCAOAOOC CATOAACCAC CCOOCTCTGA COOTOCACCC OOCACACCAO CTOOOCTCCT CCTCOCTOCA 8 a l / 5 OCTCOCTCTA TOOAOOOCCO OAOCTOOTOT TCCCAOAOOC CATOAACCAC CCOOCTCTGA COOTOCACCC OOCACACCAO CTOOOCTCCT CCTCOCTOCA
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
7 1 0 7 2 0 7 3 0 7 4 0 7 5 0 7 6 0 7 7 0 7 * 0 7 9 0 *00 2 a l / 6 ACCCCCTCAC TCTTTCTTCA OCOCCCAOCA TCOOOACCCT CTCCACTTCT ACCCCTOOOT OCTTCOOAAT COCTTCTTTO OCCACCOCTT CCAOOCAAOC 8 a l / 5 ACCCCCTCAC TCTTTCTTCA OCOCCCAOCA TCOOOACCCT CTCCACTTCT ACCCCTOOOT OCTTCOOAAT COCTTCTTTO OCCACCOCTT CCAOOCAAOC
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
8 1 0 8 2 0 8 3 0 840 850 8 6 0 8 7 0 8 8 0 8 9 0 900 2 a 1 / 6 OACOTTCCCC AOOACOOOCT OCTTTTOCAC OOOCCCTTCO CACOCAAOCC CAAOCOOATT COCACAOCCT TCTCOCCCTC OCAOCTOCTO COOCTOOAOC 8 a 1 / 5 OACOTTCCCC AOOACOOOCT OCTTTTOCAC OOOCCCTTCO CACOCAAOCC CAAOCOOATT COCACAOCCT TCTCOCCCTC OCAOCTOCTO COOCTOOAOC
. . . I . - I . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
910 920 9 3 0 940 950 9 6 0 9 7 0 9 8 0 9 9 0 1 0 0 0 2 a l / 6 OAOCCTTTGA OAAOAATCAC TACOTOOTOO OAOCCOAOCO OAAOCAOCTO OCAOOCAOCC TCAOCCTCTC COAOACOCAO OTOAAOOTOT OOTTCCAQAA 8 a 1 / 5 OAOCCTTTOA OAAOAATCAC TACOTOOTOO OAOCCOAOCO OAAOCAOCTO OCAOOCAOCC TCAOCCTCTC COAOACOCAO OTOAAOOTOT OCTTCCAOAA . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
1 0 1 0 1 0 2 0 1 0 3 0 1 0 4 0 1 0 5 0 1 0 6 0 1 0 7 0 1 0 8 0 1 0 9 0 1 1 0 0 2 a 1 / 6 TCOOAOOACA AAATACAAAC OOCAOAAOCT OOAAOAOOAA OOOCCOOAOT CTGAOCAOAA OAAOAAOGGT TCCCACCATA TCAACCOOTO OCOCATCOCC 8 a l / 5 TCOOAOOACA AAATACAAAC OOCAOAAOCT OOAAOAOOAA OOOCCOOAOT CTGAOCAOAA OAAOAAOOOT TCCCACCATA TCAACCOOTO OCOCATCOCC
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . I . . | . . . . | . . . . | . . . . | . . . . | . . . , | . . . . |
1 1 1 0 1 1 2 0 1 1 3 0 1 1 4 0 1 1 5 0 1 1 6 0 1 1 7 0 1 1 8 0 1 1 9 0 1200 2 a l / 6 ACOAAOCAOO CCAACOCOOA OOACATTOAT OTCACCTCCA ATOACTAOgg g a a c a a t c a a g a g c c c a c a a g g g c a g g g c a t t g c c t g c t g c t g g c c a a g g 8 a l / 5 ACOAAOCAOO CCAACOCOOA OOACATTOAT OTCACCTCCA ATOACTAOgg g a a c a a t c a a g a g c c c a c a a g g g c a g g g c a t t g c t t g c t g c t g g c c a a g g
- I - I - I - I . 1 1 . . . . I . . . . I . . . | . . . . | . . . . | . . . . | . . . | . . . . | . . . | . . . . | . . . | . . . . | I 1 2 1 0 1 2 2 0 1 2 3 0 12 4 0 1 2 5 0 1 2 6 0 1 2 7 0 1 2 8 0 1 2 9 0 1300 2 a l / 6 c t c c a g g g g g c c c a t g c t g g a c t c t g g c c a c t c c t t a g c c a g g c t g c a g g g a g g c c t c g a g t c a c g g c c c c a c a g g g c t t g g a g c c t g g g g c c a t c a c t g 8 a l / 5 c t c c a g g g g g c c c a t g c t g g a c t c t g g c c a c t c c t t a g c c a g g c t g c a g g g a g g c c t c g a g t c a c g g c c c c a c a g g g c t t g g a g c c t g g g g c c a t c a c t g I - I . . . . | . . . . | . . . | . I . . . . | . . . . | . . . . | . . . . | , . . . | . . . . | . . . , | . . . . | . . . . | . . . . | . . . . | . . . . | . . . | . . . . | 1 3 1 0 1 3 2 0 1 3 3 0 1340 1 3 5 0 1 3 6 0 1 3 7 0 1 3 8 0 1 3 9 0 1400 2 a l / 6 t c a g a g g g a c a a g a g a a t g g g c t g g c t g a g g c c t g g g a c t g c t c a g c c t t c t g g t g g a g a g c c t g c t g c t t g g g t g g a c c g c c c a t c a c t g c a g c c c c c c 8 a l / 5 t c a g a g g g a c a a g a g a a t g g g c t g g c t g a g g c c t g g g a c t g c t c a g c c t t c t g g t g g a g a g c c t g c t g c t t g g g t g g a c c g c c c a t c a c t g c a g c c c c c c . . . . | . . . . | . . . | . . . . | , . . . | . . . . | . . . | . . . . | . . . | . . . . | . . . | . . . . | . . . | . . . . | . . . | . . . . | , . . . | . . . . | . . . | . . . . | 1 4 1 0 1 4 2 0 1 4 3 0 14 4 0 1 4 5 0 1 4 6 0 1 4 7 0 1 4 8 0 1 4 9 0 1 5 0 0 2 a l / 6 a g c t g c t c t c c a t g t c t c t g t c t t t g t t c t g a g a c a t t t c t g t t t t a a t t t a t t t t c c a g g t c c t g c t g t a g t t c t t a g t g a c a t c c t c t c a t g t c c c c c 8 a l / 5 a g c t g c t c t c c a t g t c t c t g t c t t t g t t c t g a g a c a t t t c t g t t t t a a t t t a t t t t c c a g g t c c t g c t g t a g t t c t t a g t g a c a t c c t c t c a t g t c c c c c . I . . | - I - I . . . . 1 1 5 1 0 1 5 2 0 1 5 3 0 1540 1 5 5 0 1 5 6 0 1 5 7 0 1 5 0 0 15 9 0 2 « l / 6 c c c c c t a t g g g a a t a a t a a a a a a t c t c c c c c t t t a a a a a a a a a a a a a a a a a a ... - ... - ... c tcg ^g 8 a l / 5 c c c c c c a t g g g a a c a a c a a a a a a c c t c c c t c c c c a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a c tcgMg
Fig 3.15 Sequence alignment of clones 2al/6 and 8al/5. The mRNA is ~1.6Kb
long with a cds of 873bp (in capital bold). The PolyA^ tail consists of 18bp for clone 2al/6 and 55bp for 8al/5. Also shown are the EcoRI and Xhol cloning sites used for library construction (in italics bold).
I - I I 10 20 30 40 50 60 7 0 8 0 90 1 0 0 110 Em xl g a a c t c g g c a c g a g g c c g g c c a c a g c c c g a c c a g c a a g g g a g a g a c c c a g g a g g a c c g c c g a a g c g g c c c c c a g c a g c a c a g g c a g c c c g c g c c c g g g c g c c c g a g a g g a - I - I ■ • ■ ■ I . . . . I 120 130 1 4 0 1 50 160 1 70 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 Bmxl g c t g c c t g c c g g g c c g c g g g g a c c a c g c c c c a g g c g g c g c g c a g c g g c c c a a c c c g g c g c c a g g g c c c c c g c a c g c c c c c c g c c g c c c c g c c c c c g g c c g g c c c g c a a g g I I . . . . [ . . . . I I 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0 2 6 0 2 9 0 3 0 0 3 1 0 3 2 0 330 Em xl g c c g a g c g c g g g c g g g c g g g c g g g c g g g g g a g g c g a a g g g c g c g g g c g c g agcgcA TO TO CCAOOCTTOO TOCACACCCC OCACOOCOCC AOCOCCOOOO CACCOAOCGT r a t C A --T G C C -- G -T --- G... ...
I I I I
3 4 0 3 5 0 3 6 0 370 3 8 0 3 9 0 4 0 0 4 1 0 4 2 0 4 3 0 440 Em xl TCCCCAGAGC CCCGCTACCT CACTCCOCTT CGOCOOCCOC OACOATOTTC CAOCCCOCOO CTAAOCOOOO TTTCACCATA OAOTCCTTOO TOOCCAAOOA TOOTOOCACC
I . . , . [ . . . . 1 I a . . I
4 5 0 4 6 0 4 7 0 4 8 0 4 9 0 5 0 0 5 1 0 5 2 0 5 3 0 5 4 0 550 Em xl OOCOOOAOTC CTGOCAGCGC OOOCOCOOOC TCCCATCCAC TOOCTOTOOC COCATCAOAO OAACCOCTCA OOCCCACOOC OCTCAACTAT CCTCACCCCA OTOCOOCCOA hum an
I 5 6 0 5 7 0 5 8 0 5 9 0 6 0 0 6 1 0 6 2 0 6 3 0 6 4 0 6 5 0 660 Em xl OACOOCCTTC OTOAOTOOCT TCCCTOCCOC TOCCOCTOCO OOCOCCOOCC OCTCOCTCTA TOOAOOOCCO OAOCTOOTOT TCCCAOAOOC CATOAACCAC CCGOCTCTOA
. . . I
6 7 0 6 8 0 6 9 0 7 0 0 7 1 0 7 2 0 7 3 0 7 4 0 7 5 0 7 6 0 770 Em xl COOTOCACCC OOCACACCAO CTOOOCTCCT CCTCOCTOCA ACCCCCTCAC TCTTTCTTCA OCOCCCAOCA TCOOOACCCT CTCCACTTCT ACCCCTOOOT OCTTCOOAAT hum an G... C ... G...G... C... G ... C ...T ... C - - G C
7 8 0 7 9 0 8 0 0 8 1 0 8 2 0 8 3 0 8 4 0 8 5 0 8 6 0 8 7 0 880 Em xl COCTTCTTTO OCCACCOCTT CCAOOCAAOC OACOTTCCCC AOOACOOOCT OCTTTTOCAC OOOCCCTTCO CACOCAAOCC CAAOCOOATT COCACAOCCT TCTCOCCCTC
I I
8 9 0 9 0 0 9 1 0 9 20 9 3 0 940 9 5 0 9 6 0 9 7 0 980 990 OCAOCTOCTO COOCTOOAOC OAOCCTTTOA OAAOAATCAC TACOTOOTOO OAOCCOAOCO OAAOCAOCTO OCAOOCAOCC TCAOCCTCTC COAOACOCAO OTOAAOOTOT
I . . . . [ . . . . I . . . . 1 . . . . 1
1 0 0 0 1 0 1 0 1 0 2 0 1 0 3 0 1 0 4 0 1 0 5 0 1 0 6 0 1 0 7 0 1 0 8 0 1 0 9 0 1100 E m xl GOTTCCAOAA TCOOAOOACA AAATACAAAC OOCAOAAOCT OOAAOAOOAA OOOCCOOAOT CTGAOCAOAA OAAOAAOOOT TCCCACCATA TCAACCOOTO OCOCATCOCC
.. I ., . |
1 1 1 0 1 1 2 0 1 1 3 0 1 1 4 0 1 1 5 0 1 1 6 0 1 1 7 0 1 1 8 0 1 1 9 0 1 2 0 0 1210 Em xl ACOAAOCAOO CCAACOOOOA OOACATTOAT OTCACCTCCA ATOACTAOgg g a a c a a t c a a g a g c c c a c a a g g g c a g g g c a t t g c c t g c t g c t g g c c a a g g c t c c a g g g g g
1 2 2 0 1 2 3 0 1 2 4 0 1 2 5 0 1 2 6 0 1 2 7 0 1 2 8 0 1 2 9 0 1 3 0 0 1 3 1 0 1 3 2 0 c c c a t g c t g g a c t c t g g c c a c t c c t t a g c c a g g c t g c a g g g a g g c c t c g a g t c a c g g c c c c a c a g g g c t t g g a g c c t g g g g c c a t c a c t g t c a g a g g g a c a a g a g a a t g g I I . . . | . I 1 3 3 0 1 3 4 0 1 3 5 0 1 3 6 0 1 3 7 0 1 3 8 0 1 3 9 0 1 4 0 0 1 4 1 0 1 4 2 0 1430 g c t g g c t g a g g c c t g g g a c t g c t c a g c c t t c t g g t g g a g a g c c t g c t g c t t g g g t g g a c c g c c c a t c a c t g c a g c c c c c c a g c t g c t c t c c a t g t c t c t g t c t t t g t t c t . . . . [ . . . . I 1 4 4 0 1 4 5 0 1 4 6 0 1 4 7 0 1 4 8 0 1 4 9 0 1 5 0 0 1 5 1 0 1 5 2 0 1 5 3 0 1540 g a g a c a t t t c t g t t t t a a t t t a t t t t c c a g g t c c t g c t g t a g t t c t t a g t g a c a t c c t c t c a t g t c c c c c t t c c c t a t g g g a a t a a t a a a a a a t c t c t c t c t t t a a a a a a