In this chapter, we describe protocols to (1) assess noncovalent
interactions between L5a and plasmid DNA and (2) study the
cellular internalization of L5a/DNA complexes as a safe tool for
cellular transgenesis. This system is an effective method for DNA
delivery as it is nontoxic and simple to complex with DNA. The
potential applications may include, but not limited to, gene therapy,
transgenesis, molecular interactions, and identification of signaling
pathway and molecular mechanisms.
2 Materials
2.1 General
Molecular and Cell
Biology Reagents
1. Synthetic L5a peptide: Synthesize L5a (RRWQW) from a custom peptide synthesis service provider. Store at À20
C.
2. Plasmid DNA: The pEGFP-N1 plasmid consists of the
enhanced green fluorescent protein (EGFP) coding sequence
under the control of the immediate early cytomegalovirus
(CMV) promoter. Store at À20
C. Amplify the plasmid
using a bacterial strain of choice (like DH5α) and obtain
800 μg of purified plasmid DNA using a Plasmid DNA Maxiprep Purification kit.
3. Cell culture medium: Roswell Park Memorial Institute (RPMI)
1640 medium supplemented with 10% fetal bovine serum
(FBS) and 1Â antibiotic-antimycotic solution, such as penicillin, streptomycin, and amphotericin B. Add 50 mL FBS and
5 mL 100Â antibiotic-antimycotic solution into 445 mL
RPMI 1640 medium. Store at 4
C.
4. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 , pH 7.4. Dissolve
8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , and 0.24 g KH 2 PO 4 in
800 mL distilled water. Adjust pH to 7.4 using hydrochloric
acid or sodium hydroxide and add distilled water to 1 L. Sterilize by autoclaving and store at 4
C.
2.2 Gel
Retardation Assay
1. 10Â TBE electrophoresis buffer: 1 M Tris-borate and 20 mM
EDTA, pH 8.3. Weigh 121.1 g Tris base, 61.8 g boric acid, and
7.4 g EDTA in a 1 L glass beaker. Add distilled water to a
volume of 900 mL. Mix the solution on a magnetic stirring
plate with a stir bar until all solutes are completely dissolved,
and adjust pH to 8.3. Dilute 100 mL of this 10Â TBE buffer to
1 L to make 1Â gel running buffer. Store up to 6 months at
room temperature.
2. 0.5% agarose gel: Weigh 0.2 g SeaKem Gold agarose in a
250 mL KIMAX Erlenmeyer flask. Add 1Â gel running buffer
to a volume of 40 mL. Heat agarose solution in a microwave
until agarose is completely dissolved (see Note 1). Cool down
the agarose gel solution to about 60
C (see Note 2). Add 4 μL
L5a CPP for Intracellular Delivery of DNA
115
interactions between L5a and plasmid DNA and (2) study the
cellular internalization of L5a/DNA complexes as a safe tool for
cellular transgenesis. This system is an effective method for DNA
delivery as it is nontoxic and simple to complex with DNA. The
potential applications may include, but not limited to, gene therapy,
transgenesis, molecular interactions, and identification of signaling
pathway and molecular mechanisms.
2 Materials
2.1 General
Molecular and Cell
Biology Reagents
1. Synthetic L5a peptide: Synthesize L5a (RRWQW) from a custom peptide synthesis service provider. Store at À20
C.
2. Plasmid DNA: The pEGFP-N1 plasmid consists of the
enhanced green fluorescent protein (EGFP) coding sequence
under the control of the immediate early cytomegalovirus
(CMV) promoter. Store at À20
C. Amplify the plasmid
using a bacterial strain of choice (like DH5α) and obtain
800 μg of purified plasmid DNA using a Plasmid DNA Maxiprep Purification kit.
3. Cell culture medium: Roswell Park Memorial Institute (RPMI)
1640 medium supplemented with 10% fetal bovine serum
(FBS) and 1Â antibiotic-antimycotic solution, such as penicillin, streptomycin, and amphotericin B. Add 50 mL FBS and
5 mL 100Â antibiotic-antimycotic solution into 445 mL
RPMI 1640 medium. Store at 4
C.
4. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 , pH 7.4. Dissolve
8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , and 0.24 g KH 2 PO 4 in
800 mL distilled water. Adjust pH to 7.4 using hydrochloric
acid or sodium hydroxide and add distilled water to 1 L. Sterilize by autoclaving and store at 4
C.
2.2 Gel
Retardation Assay
1. 10Â TBE electrophoresis buffer: 1 M Tris-borate and 20 mM
EDTA, pH 8.3. Weigh 121.1 g Tris base, 61.8 g boric acid, and
7.4 g EDTA in a 1 L glass beaker. Add distilled water to a
volume of 900 mL. Mix the solution on a magnetic stirring
plate with a stir bar until all solutes are completely dissolved,
and adjust pH to 8.3. Dilute 100 mL of this 10Â TBE buffer to
1 L to make 1Â gel running buffer. Store up to 6 months at
room temperature.
2. 0.5% agarose gel: Weigh 0.2 g SeaKem Gold agarose in a
250 mL KIMAX Erlenmeyer flask. Add 1Â gel running buffer
to a volume of 40 mL. Heat agarose solution in a microwave
until agarose is completely dissolved (see Note 1). Cool down
the agarose gel solution to about 60
C (see Note 2). Add 4 μL
L5a CPP for Intracellular Delivery of DNA
115