TITLE: A recombinant  retroviral expression vector (pLXSN-HygR) based on pLXSN that confers resistance to hygromycin and pLXSN-HygR derivatives that encode EGFR, ERBB2, or ERBB3
AUTHORS: David J Riese II, Vipasha VD Dwivedi

[DESCRIPTION]
Here we describe the construction of a derivative of pLXSN, called pLXSN-HygR, that expresses a hygromycin resistance gene rather than a neomycin resistance gene.  We also describe the construction of pLXSN-HygR derivatives that express the EGFR, ERBB2, or ERBB3 genes.  Thus, these constructs are ideal for studying the functional effects of ERBB4 heterodimerization with EGFR, ERBB2, or ERBB3.

[STEPS]
SECTION: Introduction
1. Recombinant retroviruses are commonly used to direct the ectopic expression of genes in infected cells. There are two significant advantages to this approach.  Following infection of the target cell, the recombinant retroviral genome is reverse-transcribed and subsequently integrated into the host cell genome, enabling stable ectopic gene expression.  Recombinant retroviral expression vectors typically contain a drug-resistance gene, allowing the selection and maintenance of stably infected cells.
SECTION: Introduction
2. We have previously reported the construction of recombinant retroviral expression vectors based on pLXSN [1] that express the EGFR, ERBB2, ERBB3, or ERBB4 receptor tyrosine kinase genes [2, 3].  These constructs are useful for studying the role of these genes in regulating cellular proliferation, particularly in tumor cells.
SECTION: Introduction
3. However, ERBB family receptor tyrosine kinases can undergo heterodimerization, thereby diversifying the effects of ERBB receptor signaling [4, 5].  Therefore, there is a need for constructs that enable the simultaneous ectopic expression of two ERBB receptor tyrosine kinase genes.  Hence, here we describe the construction of a derivative of pLXSN, called pLXSN-HygR, that expresses a hygromycin resistance gene rather than a neomycin resistance gene.  We also describe the construction of pLXSN-HygR derivatives that express the EGFR, ERBB2, or ERBB3 genes.  Thus, these constructs are ideal for studying the functional effects of ERBB4 heterodimerization with EGFR, ERBB2, or ERBB3.
SECTION: Methods
4. Construction of pLXSN-HygR
We have previously briefly described the construction of pLXSN-HygR [6]. The plasmid pSV2-Hyg [7], which encodes the hygromycin resistance gene [8], is a generous gift from Daniel DiMaio.  
SECTION: Methods
5. The plasmid pSV2-Hyg was digested with BglII, after which the overhang was filled using the Klenow fragment of the E. Coli DNA polymerase I.  This product was digested with HindIII, releasing a 1348 bp fragment that contains the hygromycin resistance gene; the hygromycin resistance gene is 1026 bp in length (nt 2050 to 3075 of pLXSN-HygR) and encodes a 341 aa protein.  Therefore, this fragment was used as the insert for pLXSN-HygR.  The HindIII site of this fragment lies approximately 30 nt upstream of the start codon of the hygromycin resistance gene, whereas the former BglII site of this fragment lies approximately 290 nt downstream of the stop codon of the hygromycin resistance gene.
SECTION: Methods
6. The plasmid pLXSN [1, 3] (  – Figure 1) was digested with HindIII and NaeI.  Note that NaeI produces blunt ends.  The largest fragment (5071 bp) of this digestion is missing the neomycin resistance gene and was used as the vector for pLXSN-HygR.

  

SECTION: Methods
7. The pLXSN-NaeI-HindIII fragment was ligated with the pSV2-Hyg-HindIII-Blunt fragment and the ligation reaction product was electro-transformed into DH10B E. coli.  Ampicillin-resistant colonies of E. coli were expanded, and minipreps were screened for the presence and correct orientation of the hygromycin resistance gene insert.  NGS has validated this construct, and the map of the resulting sequence ( ) is shown in Figure 2.

  

SECTION: Methods
8. Construction of pLXSN-HygR-EGFR
To generate the pLXSN-HygR vector fragment, which contains the hygromycin resistance gene, we digested pLXSN-HygR (pLXSN-HygR.dna – see above for link) with AvrII and AscI (Figure 2) and isolated the 5043 bp fragment.  To generate the EGFR cDNA fragment, we digested pLXSN-EGFR ( ) [2, 3] with AscI and AvrII and isolated the 5521 bp fragment (Figure 3).

  

SECTION: Methods
9. The pLXSN-HygR-AvrII-AscI fragment was ligated with the pLXSN-EGFR-AscI-AvrII fragment and the ligation reaction product was electro-transformed into DH10B E. coli.  Ampicillin-resistant colonies of E. coli were expanded, and minipreps were screened for the correct recombinant plasmid.  NGS has validated this construct, and the map of the resulting sequence ( ) is shown in Figure 4.

  

SECTION: Methods
10. Construction of pLXSN-HygR-ERBB2
We have previously briefly described the construction of pLXSN-HygR-ERBB2 [9].  To generate the pLXSN-HygR vector fragment, which contains the hygromycin resistance gene, we digested pLXSN-HygR (pLXSN-HygR.dna – see above for link) with HindIII and XmnI (Figure 2) and isolated the 4017 bp fragment.  To generate the ERBB2 cDNA fragment, we digested pLXSN-ERBB2 ( ) [2, 3] with XmnI and HindIII and isolated the 6469 bp fragment (Figure 5).

  

SECTION: Methods
11. The pLXSN-HygR-HindIII-XmnI fragment was ligated with the pLXSN-ERBB2-XmnI-HindIII fragment and the ligation reaction product was electro-transformed into DH10B E. coli.  Ampicillin-resistant colonies of E. coli were expanded, and minipreps were screened for the correct recombinant plasmid.  NGS has validated this construct, and the map of the resulting sequence ( ) is shown in Figure 6.

  

SECTION: Methods
12. Construction of pLXSN-HygR-ERBB3
We have previously briefly described the construction of pLXSN-HygR-ERBB3 [9].  To generate the pLXSN-HygR vector fragment, which contains the hygromycin resistance gene, we digested pLXSN-HygR (pLXSN-HygR.dna – see above for link) with StuI and AseI (Figure 2) and isolated the 3607 bp fragment.  To generate the ERBB3 cDNA fragment, we digested pLXSN-ERBB3 ( ) [2, 3] with AseI and StuI and isolated the 7116 bp fragment (Figure 7).
 
  

SECTION: Methods
13. The pLXSN-HygR-StuI-AseI fragment was ligated with the pLXSN-ERBB3-AseI-StuI fragment and the ligation reaction product was electro-transformed into DH10B E. coli.  Ampicillin-resistant colonies of E. coli were expanded, and minipreps were screened for the correct recombinant plasmid.  NGS has validated this construct, and the map of the resulting sequence ( ) is shown in Figure 8.

  

SECTION: Conclusion
14. Here we describe the construction of a recombinant retroviral vector (pLXSN-HygR) that carries the hygromycin resistance gene instead of the neomycin resistance gene, as well as derivatives that also carry the EGFR, ERBB2, or ERBB3 cDNAs (pLXSN-HygR-EGFR, pLXSN-HygR-ERBB2, and pLXSN-HygR-ERBB3, respectively).  These constructs, together with the recombinant retroviral vector that carries the neomycin resistance gene and the ERBB4 cDNA (pLXSN-ERBB4) [2, 3], will enable us to generate bi-recombinant cell lines to study the effects of ERBB4 receptor heterodimerization with the other ERBB family receptors.
SECTION: References
15. 
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2.  Riese, D.J., 2nd, et al., The cellular response to neuregulins is governed by complex interactions of the erbB receptor family. Mol Cell Biol, 1995. 15(10): p. 5770-6.

3.  Riese II, D.J. Recombinant retroviral expression vectors based on LXSN that encode EGFR, ERBB2, ERBB3, and ERBB4. Protocols.io 2023  [Accessed August 21, 2023]; Available from: https://dx.doi.org/10.17504/protocols.io.261gedd97v47/v1.

4.  Riese, D.J., 2nd and D.F. Stern, Specificity within the EGF family/ErbB receptor family signaling network. Bioessays, 1998. 20(1): p. 41-8.

5.  Lucas, L.M., et al., The Yin and Yang of ERBB4: Tumor Suppressor and Oncoprotein. Pharmacol Rev, 2022. 74(1): p. 18-47.

6.  Riese, D.J., 2nd and D. DiMaio, An intact PDGF signaling pathway is required for efficient growth transformation of mouse C127 cells by the bovine papillomavirus E5 protein. Oncogene, 1995. 10(7): p. 1431-9.

7.  Lin, B.F. and B. Shane, Expression of Escherichia coli folylpolyglutamate synthetase in the Chinese hamster ovary cell mitochondrion. J Biol Chem, 1994. 269(13): p. 9705-13.

8.  Bernard, H.U., G. Krammer, and W.G. Rowekamp, Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture. Exp Cell Res, 1985. 158(1): p. 237-43.

9.  Feroz, K., E. Williams, and D.J. Riese, 2nd, ErbB2 and ErbB3 do not quantitatively modulate ligand-induced ErbB4 tyrosine phosphorylation. Cell Signal, 2002. 14(9): p. 793-8.