Rescue of an enterotropic Newcastle disease virus strain ZM10 from cloned cDNA and stable expressing an inserted foreign gene

Table 1 Primer sequences of the construction of ZM10 full-length cDNA clone

Primer	Primer sequence	Primer name
1^a	ACCAAACAGAGAATCCGTAAG	F1 F
2^a	CAAGAGATATGACAGTTAAAACG	F1 R
3^a	CTGTCATATCTCTTGTATGTGGTATACTTAGCCTGG	F2 F
4^a	ATCCAGTCTATGTTGGAGATTCCCAGCTG	F2 R
5^a	CAACATAGACTGGATGACGGCATAACTCAGATGAC	F3 F
6^a	ACCAAACAAAGATTTGGTGAATGACAAGAC	F3 R
7^b	ACAACCCTGTCCTGCTTCCTCTG	RFP-F
8^b	CGCTCGGGGTGTTGGACCTTGGG	RFP-R
9^b	CTGTCATATCTCTTGGGCCGGCATGGTCCCAGCCTCC	F1 Vec F
10^c	GATTCTCTGTTTGGTCCCTATAGTGAGTCGTATTAGCG	F1 Vec R
11 ^c	CAACATAGACTGGATGGCCGGCATGGTCCCAGCCTCC	F2 Vec F
12^c	AAATCTTTGTTTGGTGGCCGGCATGGTCCCAGCCTCC	F3 Vec F
13^c	CCAACACCCCGAGCGCAACTCTCCAAGCGGCAATC	Vec-RFP-F
14^c	GCAGGACAGGGTTGTAGCGAGAGAGGTAACGATTAG	Vec-RFP-R
15^d	AAACACGATAATACCATGTCTTCTGTATTCGATGAG	NP F
16^d	GTGGATCGGATCTTATCAGTACCCCCAGTCGGTGTC	NP R
17^d	AAACACGATAATACCATGGAGATGGCCACCTTTACAG	P F
18^d	GTGGATCGGATCTTATTAGCCATTCAGTGCAAGGCGC	P R
19^d	AAACACGATAATACCATGGCGAGCTCCGGTCCCGAG	L F
20^d	GTGGATCGGATCTTATTAAGAGTCACAGTTACTGTAG	L R
21^d	TAAGATCCGATCCACTAGTTCTAG	Vet up
22^d	GGTATTATCGTGTTTTTCAAAGGAAAACC	Vet down
23^e	TGGACCCTGGTTGTGCC	RFP dect F
24^e	GGCTTCTTCATTCCCAACC	RFP dect R

All the primers were labeled with a superscript lowercase letter to better illustrate them. Nucleotides shown in bold letters represent homology sequences with a vector backbone, which were used to facilitate the RE independent cloning using the In-Fusion® PCR Cloning Kit (Clontech). ^aPrimers 1–6 were used to RT-PCR amplify the Fragment 1, Fragment 2, and Fragment3 genes from the viral RNA of NDV ZM10 strain. ^bPrimers 7–8 were used to amplify the RFP genes from LS-RFP plamid. ^cPrimers 9–14 were used to amplify or linearize the subclone vectors and the vector for RFP gene. ^dPrimers 15–22 were used to generate the helper plasmids of NDV strain ZM10. ^ePrimers 23–24 were used to amplify the insert part and a part of NDV vector gene

ISSN: 1472-6750