Xenopus Resources Available

Resources Available

Table 1:Xenopus Lymphocyte surface markers detectable with currently available mAbs

Xenopus markers (mAbs)	Expression pattern
CD8 (AM22, F17)	Larval and adult thymocytes (70-80%) and T-cells (about 20% of splenocytes). All lymphoid tumor lines
CTX (X71, 1S9.2)	Larval and adult thymocytes (60-70%)no consistent expression in periphery. All lymphoid tumor lines and gut epithelial tissue.
XT1 (XT1)	Most, but not all, larval and adult T-cells; earliest marker of thymocytes. All lymphoid tumor lines
MHC class I (TB17)	Ubiquitous in adult; all lymphopoietic lineages. Not expressed until metamorphosis
MHC class II (AM20, 14A2)	Thymocytes, B and T-cells (99% of spleen lymphocytes), only B-cells in larvae
CD5 (2B1)	Thymocytes (>95%), T-cells and some PMA-activated IgM+ B cells. All lymphoid tumor lines
NK-like (1F8)	Non-B and non-T, peripheral lymphoid cells.
RC47	Leukocyte lineage from very early stage. Thymic cortex and medulla (>90% of total thymocytes)
IgM (10A9)	Larval and adult B cells.
IgY (11D5)	Some larval and adult B cells.
IgX (410D9)	Some larval and adult B cells, especially in the gut.

Table 2: Some available cDNA encoding immunologically-relevant X. laevis genes

Immune System Characteristics	Larva (Ancestral-like system) Adult (Mammalian-like system)
CTX	Thymus, all lymphoid tumor lines and gut epithelial tissue.
TCR α, β, γ	thymus, circulating T-cells
BCR μ, γ, ξ, L	B cells
MHC class I	ubiquitous in adult
Non-classical class Ib	more restricted tissue distribution for some of them
MHC class II	thymus, splenic lymphocytes
Rag 1 & 2	mainly thymus, at lower level in bone marrow, kidney, spleen and liver
TdT	mainly thymus of old larvae and adult, less in spleen
Ikaros	mainly thymus
GATA III	thymus and T-cells
LMP7	Thymus, spleen
Hsps (gp96, hsp70)	Ubiquitousely expressed,

Table 3: Currently available X. laevis MHC-defined stains and clones

	Name, (MHC gentotype)
Partially inbred, MHC homozygous strains	F, J,
Isogenetic leavis/gilli (LG) clones with identical heterozygous (a/c) MHC genotype	LG-6, LG-7, LG-15
MHC-disparate LG isogenetic clones	LG-3 (b/d), LG-5 (b/c)

Table 4: Available cloned thymic tumor cell lines

Name of Tumor Cell Line	BB7	ff-2	15/0
Genetic background of initial tumor-bearing host	Partially inbred MHC homozygous ff strain		LG-15 (MHC a/c) isogenetic clone
T-cell surface markers (CD8, CD5, pan T-cell)	+++	+++	+++
Ig mRNA	-	+	+
Ig protein	-	-	-
MHC class I mRNA and protein	-	+	-
MHC class II mRNA and protein	-	-	-
Tumorigenicity in syngeneic
Larvae	-	+	++
Adult

List of available genomic and cDNA libraries

X. laevis cDNA libraries
Old libraries in λZAPII vector
ST:	Outbred adult spleen + thymus (constructed by L. Du Pasquier, Basel Switzerland))
LG7Ad:	Adult from the clone LG7 immunized with DNP-KLH + CFA (constructed L. Du Pasquier)
LG7 Tadp:	LG7 pre-metamorphic larvae immunized DNP-KLH + alum (constructed by M. Wilson, Jakson MS)
SMART cDNA libraries (λZAP-express, constructed by S. Guselnikov and A. Taranin, Novosibirsk, Russia)
T:	Outbred larval spleen + thymus + liver. 7x10⁵ independent clones, titer 6x10⁹
S:	Outbred adult spleen. 2x10⁶ independent clones, titer 1x10⁹
NK:	NK cells, 1F8+ purified from outbred adult splenocytes. 3x10⁶ independent clones, titer 1x1010
T:	CD8 NK/T cells (AM22+/1F8+) purified from outbred splenocytes. 5x10⁵ independent clones, titer 4x10⁹
Arrayed EST cDNA libraries (constructed by Express Genomics, Inc)
	Outbred adult thymus
	Outbred adult splenocytes activated for 4 days with PHA
X. tropicalis cDNA libraries (λZAP-express)
Trop:	Adults spleen + thymus + intestine. Titer 5x10¹⁰

Table 5: Usual Primer Pairs For RT-PCR

Activation-induced cytosine-deaminase (AID):
Forward	5’-TCA CGA CCC CCA TAG GAA CTA C-3’
Reverse	5’-GGA TTT AGG AGA CTT TGC CTC AAG-3’
TM	58ºC; product length: 450 bp (span intron)
β-actin:
Forward	5’-GGT GTC ATG GTT GGA ATG G-3’
Reverse	5’-TGG GTT ACA CCA TCA CCT GAG-3’
TM	56ºC; product length: 359 bp
β2-microglobulin
Forward	5’-CCG GTG GTC AAG GTT TAC ACT G-3’
Reverse	5’-AGC TGT GAA AGA TTA AAC CCT-3’
TM	57ºC; product length: 800, 950 bp (2 bands correspond to the 2 X. laevis genes; primers also span an intron)
α2-macroglobulin receptor CD91:
Forward	5’-AAT GGA GAC ATC ATC TGC TCG TG-3’
Reverse	5’- CAG GGT CGC ATA GAC TGG ATT G-3’
TM	56ºC; product length: 500 bp (span intron)
Chytrid Fungus ribosomal RNA Intervening Transcribed Sequence (Batrachochytrium dendrobatidis ITS)
Forward	5’- CCT TGA TAT AAT ACA GTG TGC CAT ATG TC-3’
Reverse	5’- AGC CAA GAG ATC CGT TGT CAA A-3’
TM	60ºC; product length: 250 bp (span an intron)
CTX:
Forward	5’-GCA GCA GCG GTA ATC GGA G-3’ (position 214)
Reverse	5’-CTC AGC ATG GTC ATG GAA TTG-3’ (position 287)
TM	56ºC; product length: 225 bp (1.3 kb if genomic DNA amplified)
Elongation factor α1 (Ef-α1):
Forward	5’-CCT GAA TCA CCC AGG CCA GAT TGG TG-3’
Reverse	5’-GAG GGT AGT CTG AGA AGC TCT CCA CG-3’
TM	56ºC; product length 223 bp
Frog virus 3 Immediate early protein (IE-FV3):
Forward	5’- ATG ATC CAA GCC TAC CTG TGC-3’
Reverse	5’- AAA TGT CCT AAT CTA TAC ACC-3’
TM	51.5ºC; product length 420 bp
Frog virus 3 major capsid protein (MCP):
Forward	5’- GAC TTG GCC ACT TAT GAC -3’
Reverse	5’- GTC TCT GGA GAA GAA GAA-3’
TM	55ºC; product length 520 bp. Remark: primers work also with ATV
Gp96:
Forward	5’- GTTGTAAGCAGGGAAGAGG-3’
Reverse	5’-TGTCTCAGTCTTGCTGCTCCACAC-3’
TM	56ºC; product length 500 bp
HSC-70
Forward	5’-TGG AGT AGA AAG GAA TGT ACT GAT C-3’
Reverse	5’-AAC TTC ATT GCA TTT CTC CAG-3’
TM	58ºC; product length: 747 bp
HSP70
Forward	5’-AGA CTG ATC GGG AGA AAG TTC-3’
Reverse	5’-CTT TTC CAC AGG TTC CAA GGT ACC C-3’
TM	58ºC; product length: 747 bp
Immunoglobulin heavy chain μ, constant domain (IgH-Cμ):
Forward	5’- GCA ATG CCA AAC ACC TGG-3’
Reverse	5’- TCT TCT CTT GTA AGG AAC CCG-3’
TM	58ºC; product length 350 bp
Immunoglobulin heavy chain γ, constant domain (IgH-Cγ):
Forward	5’- ATC CAG CAG CCA ACT CAC CAT C -3’
Reverse	5’- AAG GTG GCA GCA AGG TGA TTG -3’
TM	58ºC; product length 720 bp
Interleukin 1β (IL-1β):
Forward	5’- AAC AGA AGA TGG CCA AGA CTC-3’
Reverse	5’- ATG CAA CCG ATT CAA AGC TG-3’
TM	55ºC; product length:152 bp. (span an intron)
Low mass protein 7 (LMP7):
Forward	5’- CAG AAT TCG CNG AYT CYC ART AYT GG -3’
Reverse	5’- CAG AAT TCC CAN CCR TCY TCY TTC AT-3’
TM	53ºC; product length 400 bp
Major histocompatibility antigen class I (MHC class I):
Forward	5’-GAGTTCTTTGCCCTGGATACA-3’ (Lineage A α2-specific)
Forward	5’-GAGTTCATAGCGCTGGATACA-3’(Lineage B α2-specific)
Reverse	5’-CACAATAAGTTTCTCCTTCAGG-3’(Lineage A, and B specific)
TM	59ºC; product length: 450 bp
Major histocompatibility antigen class IIα (MHC class IIα):
Forward	5’-TCAAAGTCAGTGGTTTGGACG -3’
Reverse	5’-GTGTAAATATCATGTTCATTTGGT-3’
TM	58ºC; product length: 390 bp
Major histocompatibility antigen class class IIΒ (MHC class IIΒ):
Forward	5’-ACGGCACCGACAATGTCAGG -3’
Reverse	5’-TTAATGGTTGTCTCCAGCATC-3’
TM	58ºC; product length: 450 bp
Recombination activating gene 1 (Rag 1):
Forward	5’- GCG CCA AGA ATCTGT GTC ACT-3’
Reverse	5’- GAT AGC TCC GGT TCT GCT GAT-3’
TM	58ºC; product length: 446 bp
Recombination activating gene 2 (Rag 2):
Forward	5’- GAG CAC AGA GGG AAG AAC AG-3’
Reverse	5’- (GCA CAC AGC TGG ATG CCT AA-3’
TM	56ºC; product length: 298 bp
Terminal deoxynucleotidal transferase (TdT):
Forward	5’- ATC CAC TGA GCC AAT-3’
Reverse	5’- CCT TCA TCG CTG TTA-3’
TM	56ºC; product length: 616 bp