Figure S1. Molecular cloning of goat Nanog gene: a. PCR amplification of gNanog cDNA
amplified 1049bp long PCR product. b. Cloning and transformation produced large number of
gNanog positive colonies. c. PCR product (1049bp) amplified from of plasmid isolated from
positive clones for confirmation of Nanog Gene inserts.
Figure S2. cDNA sequence of goat Nanog gene. a. cDNA sequence containing ORF of
903bp(grey coloured area). b. ORF contains Hox domain coding region (green colour).
Figure S3:
Figure: S3 nucleotide sequence alignment of goat with cattle, buffalo, Pig, Human, and
Mouse.
Figure S4
Figure S4. Phylogenetic tree and percentage identity. A. phylogenetic tree among goat,
cattle, buffalo, pig, human and mouse shows goat is evolutionary more closely related
to cattle than other species. B. goat Nanog sequence is 96.2% identical to cattle and
having 3.9 divergence to cattle which also shows the closeness of goat and cattle.
Figure S5
Figure S5: Transfected CHO-K1(a,b,c,d) expressing Nanog gene with GFP as a reporter gene after 3 day
of Transfection. Transfected CHO-K1 (e,f,g,h)expressing Nanog gene with GFP as a reporter gene
after 4th passage of Transfection . Transfected CHO-K1 (i,j,k,l) expressing Nanog gene with GFP
as a reporter gene after 7th passage of Transfection
Figure S6: a. pAcGFP1-N1 expression vector. b. PCR product amplified from the plasmids
isolated from recombinant pAcGFP_Nanog positive clones.
Table 1: Different primers used for cDNA amplification and expression cloning.
Primes
Sequence
Accession no.
<5’ ---------------------------------- 3’>
Nanog_A
ACCCGGAGATCTTCACCTTT
NM_001025344
Nanog_AS
CCCATACAATAGCCGTCACA
NM_001025344
Nanog_Nhe1
GAGTTCGCTAGCATGGGGAGTGTGGACCCAGCTTGTCC
JQ801747
Nanog_Sal1
CAGTGTCGACCAAATCTTCAGGCTGTATGTTGAGAGG
JQ801747
Nanog-f-RT
TGGAGCAATCAGACCTGGAACAGT
JQ801747
Nanog-r-RT
CAGTGATTTGCTGCTGGGAACTGA
JQ801747
Nanao-f-Topo
CACCGTAATGGGGAGTGTGGACCCAGCTT
JQ801747
Nanao-r-Topo
TTACAAATCTTCAGGCTGTATGTTGAGAG
JQ801747
GAPDH_F
CCAACGTGTCTGTTGTGGATCTGA
NM_001034034
GAPDH_R
GAGCTTGACAAAGTGGTCGTTGAG
NM_001034034
Table 2: Recombination reaction as per the manufacturer’s instruction.
Sr no
Reagents
Chemical Transformation
1
Fresh PCR product
3ul
2
Salt solution
1ul
3
Nuclease free Water
1ul
4
Topo vector
1ul
Total volume
6 ul
Table 2: Recombination Reaction for lentiviral cloning. Topo vector: denotes lentiviral vector
pEnter/D-topo.
Table 3: Table 3: Transformation of Recombinant Nanog lentiviral expression constructs.
Component
Entry clone
Destination vector (150 ng/ul)
TE buffer, pH 8.0
Sample volume
4ul (50 ng/ul)
1ul
To 8ul
Incubated for 5 min at room temperature and used for transformation.
Table 3: Transformation of Recombinant Nanog lentiviral expression constructs.