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APPENDIX

7.1 EMBL/GenBank™ accession numbers (nucleotide sequence)

PDGF
hPDGF-A, genomic	S51624 (part one)
hPDGF-A, genomic	S50869 (part two)
hPDGF-B, cDNA	M12783; M16288

VEGF
hVEGF165, cDNA	M32977
hVEGF, genomic	M63971 (exon 1)
hVEGF, genomic	M63972 (exon 2)
hVEGF, genomic	M63973 (exon 3)
hVEGF, genomic	M63974 (exon 4)
hVEGF, genomic	M63975 (exon 5)
hVEGF, genomic	M63976 (exon 6)
hVEGF, genomic	M63977 (exon 7)
hVEGF, genomic	M63978 (exon 8)
mVEGF, genomic	(alternatively spliced exon 6)
mVEGF promoter	U41383

PlGF
hPlGF149, cDNA	X54936
rPlGF132/135, cDNA	L40030

VEGF-B
hVEGF-B167, cDNA	U48801
hVEGF-B186, cDNA	U52819
mVEGF-B186, cDNA	U52820
hVRF186, cDNA	U43368
hVRF167, cDNA	U43369
mVRF186, cDNA	U43836
mVRF167, cDNA	U43837

VEGF-C
hVEGF-C, cDNA	X94216
mVEGF-C, cDNA	U73620
hVRP, cDNA	U43142

FLT1
hFLT1, cDNA	X51602
sFLT1, cDNA	U01134

KDR/FLK1
FLK1, cDNA	X59397
KDR, cDNA	L04947

FLT4
hFLT4s, cDNA	U43143
hFLT4l, cDNA	X68203; X69878; S59182
hFLT4l, cDNA	S66407 (alternatively spliced 3’-end)
mFLT4, cDNA	L07296

7.2 Genomic structures and mRNA splice variants of VEGF, VEGF-B and VEGF-C

Figure 16. Genomic organization of the human VEGF gene and mRNA splice variants
Legend: squares: exons (empty=untranslated sequence, filled=translated sequence); lines: introns; numbers above exons: exon length (bp); numbers under introns in italics: intron length (kb) according to Tischer (Tischer et al., 1989), in brackets according to Houck (Houck et al., 1991); scale: 1 cm exon=83 bp, 1 cm intron=2 kb.
* The lengths given for the 3'-UTR refer to the polyadenylation signals identified in the rat VEGF gene (Levy et al., 1995), the length of the major transcript in both human and rat (approximately 3.7 kb; Goldberg and Schneider, 1994) suggests for the major human mRNA a 3'-UTR of approximately 2.1 kb.

Figure 17. Genomic organization of the murine VEGF gene and mRNA splice variants
Legend: squares: exons (empty=untranslated sequence, filled=translated sequence); lines: introns; numbers above exons: exon length (bp) according to Breier (Breier et al., 1992); numbers under introns: intron length (kb) according to Shima (Shima et al., 1996); scale: 1 cm exon=83 bp, 1 cm intron=2 kb.
* The length given for the 3'-UTR refers to one identified site of transcription termination (Shima et al., 1996); however mRNA transcripts vary in length between 2.7 kb and 4 kb, indicating that upstream at least one alternative termination site is present.

Figure 18. Genomic organization of the human VEGF-B gene and mRNA splice variants
Legend: squares: exons (empty=untranslated sequence, filled=translated sequence); lines: introns; numbers above exons: exon length (bp); numbers under introns in italics: intron length (kb); scale: 1 cm exon=83 bp, 1 cm intron=1 kb (data according to Olofsson et al., 1996b).
* The 3'-UTR comprises 381 bp (personal communication by Suvi Taira) and based on the length of the mature mRNA for VEGF-B186 (approximately 1.4 kb; Olofsson et al., 1996b) the transcriptional start site can be expected about 400 bp upstream of the translation initiation ATG.

Figure 19. Genomic organization of the murine VEGF-B gene and mRNA splice variants
Legend: squares: exons (empty=untranslated sequence, filled=translated sequence); lines: introns; numbers above exons: exon length (bp); numbers under introns in italics: intron length (kb); scale: 1 cm exon=100 bp, 1 cm intron=1 kb (data according to Olofsson et al., 1996b).
* Length of 3'-UTR and the transcriptional start site seem to be similar for the murine and human gene (personal communication by Suvi Taira) and the lengths refer to the human gene.

Figure 20. Genomic organization of the human VEGF-C gene and mRNA splice variants
Legend: squares: exons (empty=untranslated sequence, filled=translated sequence); lines: introns; numbers above exons: exon length (bp) according to Dmitry Chilov (personal communication); numbers under introns in italics: intron length (kb) according to Dmitry Chilov (personal communication); scale: 1 cm exon=100 bp, 1 cm intron=1 kb, if the intron size was not or only approximately known, the drawing was based on the size of the corresponding mouse gene intron according to Suvi Taira (personal communication).
* The lengths given for the 3'-UTR refers to the polyadenylation signal present in the first VEGF-C cDNA clone (Joukov et al., 1996). The two shorter forms of VEGF-C are hypothetical, mRNA species of corresponding lengths are described (Lee et al., 1996), but it is unknown whether they are translated.

Figure 21. Genomic organization of the murine VEGF-C gene and mRNA splice variants
Legend: squares: exons (empty=untranslated sequence, filled=translated sequence); lines: introns; numbers above exons: exon length (bp) according to Suvi Taira (personal communication); numbers under introns in italics: intron length (kb) according to Suvi Taira (personal communication); scale: 1 cm exon=100 bp, 1 cm intron=1 kb.
* The length given for the 3'-UTR refers to the known genomic clone covering the 3'-flanking region with the polyadenylation signal, the length of the 5'-UTR is deduced on basis of the major murine transcript (approximately 2.4 kb; Suvi Taira, personal communication).

7.3 Exon/intron borders of VEGF, VEGF-B and VEGF-C genes

Figure 22. Exon/intron borders for human and murine VEGF
E=exon; upper case characters=exonic sequence; lower case characters=intronic sequence; the length of the translated parts of the exons is indicated; intronic sequence length in brackets refers to the intron length given by Houck et al., which differs for the introns 4, 5, 6 and 7 from the data given by Tischer et al. (Sources: Breier et al., 1992; Houck et al., 1991; Shima et al., 1996; Tischer et al., 1989).

Figure 23. Exon/intron borders for human and murine VEGF-B
E=exon; upper case characters=exonic sequence; lower case characters=intronic sequence; the length of the translated parts of the exons is indicated (Source: Olofsson et al., 1996b).

Figure 24. Exon/intron borders for human and murine VEGF-C
E=exon; upper case characters=exonic sequence; lower case characters=intronic sequence; the length of the translated parts of the exons is indicated. * The shorter forms of VEGF-C are hypothetical, mRNA species of corresponding lengths are described for humans (Sources: Eola Kukk, personal communication; Lee et al., 1996).

7.4 Plasmid maps

Figure 25. K14-VEGF-B167

Figure 26. K14-VEGF-B186

Figure 27. K14-VEGF-C-FL

Figure 31. RIP-VEGF-B167

Figure 32. RIP-VEGF-C-FL

Figure 33. pFB1-VEGF-B167

Figure 34. VEGF-B167-X3-pCRII

Figure 35. pVT-Bac-melSP-VEGF-B167

Figure 36. pFB1-melSP-VEGF-B167

Figure 37. pFB1-CHIS-VEGF-B186

Figure 38. pFB1-melSP-CHIS-VEGF-B186

Figure 39. pFB1-VEGF-C

Figure 40. VEGF-C-X3-pCI-neo

Figure 41. pVT-melSP-N

-VEGF-C

Figure 42. pFB1-melSP-N

-VEGF-C

Figure 43. pFB1-VEGF-C-SSV (short splice variant)

Figure 44. pFB1-melSP-N

-VEGF-C-SSV (short splice variant)

Figure 45. pFB1-C

-C1HIS-VEGF-C
VEGF-C with C-term. H6-tag (CMSKLHHHHHH*=C1HIS), C-term. deletion (

= nt 997 - end, corresponding

= aa 216 - end of prepropeptide) in pFASTBAC1, blunted and SphI-cut PCR product (forward hybrid primer:5'-GGAATTCACAGAAGAGACTATAAA-3' [SBL-internal no 4047]and reverse hybrid primer: 5'-GGAATTCAATGATGATGATGGTGATGCAGTTTAGACATGC-3'[SBL-internal no 4048] applied to p1.pcDNAI [Vladimir Joukov]) ligated to HindIII(blunt)/SphI-opened pFB1-VEGF-C

Figure 46. pFB1-melSP-

C-C1HIS-VEGF-C
VEGF-C with C-term. H6-tag (CMSKLHHHHHH*=C1HIS), C- &N-term. deletions (

= start - nt 657, corresponding

= start- aa 102 of prepropeptide and

= nt 997 - end, corresponding

= aa 216 -end of prepropeptide) and melittin signal peptide in pFASTBAC1, T4 polymerase-blunted and SphI-cut PCR-fragment (as described for pFB1-C

-C1HIS-VEGF-C) ligated to HindIII-opened, blunted and subsequently SphI-cut pFB1-melSP-N

-VEGF-C

Figure 47. pFB1-C

-C2HIS-VEGF-C
VEGF-C with C-term. H6-Tag (VHSIIHHHHHH*=C2HIS) and C-term. deletion (

= nt 1027 - end, corresponding

= aa 226 - end of prepropeptide), AccI/NotI-fragment from VC

CHIS-pALTER (Vladimir Joukov) ligated to partial AccI/NotI-opened pFB1-VEGF-C

Figure 48. pFB1-melSP-

C-C2HIS-VEGF-C
VEGF-C with C-term. H6-Tag (VHSIIHHHHHH*=C2HIS), C- & N-term. deletion (

= start - nt 657, corresponding

= start - aa 102 and

= nt 1027 - end, corresponding

= aa 226 - end of prepropeptide) and melittin signal peptide in pFASTBAC1, NotI/NruI-fragment from VC

CHIS-pALTER (Vladimir Joukov) cloned into NotI/NruI-opened pFB1-melSP-N

-VEGF-C

Figure 49. pFB1-

C-C2HIS-VEGF-C
VEGF-C with C-term. H6-Tag (VHSIIHHHHHH*=C2HIS), N- & C-term. deletion (

= nt 445 - 658, corresponding

= aa 32 - 102 of prepropeptide), NotI/EcoRI-fragment from VC

CHIS-pALTER (Vladimir Joukov) cloned into NotI/EcoRI-opened pFASTBAC1

Figure 50. pFB1-C3HIS-VEGF-C
VEGF-C with C-term. H6-tag (KRPQMSHHHHHH*), NotI/EcoRI-fragment from VEGF-C-CHIS-pALTER (Vladimir Joukov) ligated to NotI/EcoRI-opened pFASTBAC1

Figure 51. pFB1-melSP-C3HIS-VEGF-C
VEGF-C with C-term. H6-tag (KRPQMSHHHHHH*), NotI/EcoNI-fragment from VEGF-C-CHIS-pALTER (Vladimir Joukov) ligated to NotI/EcoNI-opened pFB1-melSP-N

-VEGF-C

Figure 52. pFB1-NHIS-VEGF-C
VEGF-C with N-term. H6-tag (PAAAAAHHHHH HESGLDL) and one aa-deletion (D=nt 445 - 447, corresponding D=aa 32 of prepropeptide), EcoRI/ NotI-fragment from VC-NHIS-pALTER (Vladimir Joukov) ligated to EcoRI/NotI-opened pFASTBAC1

7.5 Characterization of recombinant proteins

Figure 53. Schematic representation of recombinant baculoviral VEGF-C proteins (selection)
Legend: The upper panel shows, how different parts of the protein contribute to its molecular weight, the calculated molecular weight of each protein is given for the secreted propeptide (including N-glycosylation at all three potential sites)

Figure 54. VEGF165
IP/Western of HF cell transfection supernatants and lysates, clones 11-14

Figure 55. VEGF-B167
IP/Western of Sf9 cell transfection supernatants and lysates, clones 21-24

Figure 56. VEGF-B167 (clone 23)
IP/Western of Sf9 cell lysates and conditioned medium after 3. amplification

Figure 57. No release of VEGF-B167 upon heparin treatment
IP/Western, Sf9 cells, clone 23

Figure 58. melSP-VEGF-B167
IP/Western of HF cell transfection supernatants and lysates, clones 1-7

Figure 59. melSP-CHIS-VEGF-B186
IP- and straight Western of medium conditioned by HF or Sf9 cells during 1. amplification

Figure 60. Affinity purification of melSP-CHIS-VEGF-B186 from medium conditioned by HF cells
Silver staining of 15% SDS-PAGE, clone 3

Figure 61. Metabolic labelling of VEGF-C
Sf9 cells, lysate, clone 34/3 after 3. amplification

Figure 62. VEGF-C harvested 7 d p.i.
Straight Western of Sf9 cell lysates after 2. amplification, clones 32/1-32/5 and 34/1-34/5

Figure 63. VEGF-C produced by Sf9 cells
Straight Western of cell lysates and conditioned medium; clones 32/1, 32/5, 34/2 and 34/3 after 3. amplification

Figure 64. VEGF-C produced by HF cells
Straight Western of cell lysates and conditioned medium; clones 32/1, 32/5, 34/2 and 34/3 after 3. amplification

Figure 65. melSP-N

-VEGF-C
Straight Western of HF cell lysates and conditioned medium, clones 1-7 after 2. amplification

Figure 66. melSP-

C-C1HIS-VEGF-C and C

-C1HIS-VEGF-C
Straight Western of HF cell transfection supernatant, clones 1-5

Figure 67. Differently H6-tagged VEGF-C, reducing SDS-PAGE
Straight Western of Sf9 cell transfection supernatant

Figure 68. Differently H6-tagged VEGF-C, non-reducing SDS-PAGE
Straight Western of HF cell conditioned medium, after 3. amplification

Figure 69. Short splice variant of VEGF-C and melSP-N

-VEGF-C
Straight Western of medium conditioned by HF cells, after 2. amplification

Figure 70. FLT4 stimulation with transfection supernatant
Stimulation with 1:10 dilutions of Sf9 cell transfection supernatants and medium conditioned by Sf9 cells infected with plaque-purified clones 34/2 and 34/5, VEGF-C

Figure 71. FLT4 and KDR stimulation with melSP-

C-C1HIS-VEGF-C
Stimulation with 1:2 dilutions of medium conditioned by HF cells, clone 4

Figure 72. Stimulation of FLT4, comparison between Sf9 and HF cells
Stimulation with 1:5 dilutions of conditioned medium

Figure 73. Titration of FLT4 stimulation with melSP-

C-VEGF-C
Stimulation with medium conditioned by Sf9 cells, clone 4

Figure 74. FLT4 stimulation with differently H6-tagged VEGF-C
Stimulation with 1:2 dilutions of Sf9 cell transfection supernatant

Figure 75. Affinity purification of melSP-

C-C1HIS-VEGF-C
Silver staining of 15% SDS-PAGE, purification from medium conditioned by HF cells, clone 1

Figure 76. Comparison between Silver and Coomassie Brilliant Blue staining
15% SDS-PAGE, melSP-

C-C1HIS-VEGF-C, clone 1

7.6 Pedigrees of transgenic mice

Figure 77.

MHC-VEGF-B167 transgenic mice

Figure 78. K14-VEGF-C-FL transgenic mice

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