Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 37

2 Materials

The selection of basic materials and proper preparation of reagents
required for lentiviral production is critical for success. Indeed for
many of the materials it is recommended that these specifi c vendors
and catalog numbers be utilized. A complete list of stock reagents
needed, including catalog number and vendor name, as well as
equipment required is provided in Tables 3 and 4 . Cell culture
conditions for all procedures in this chapter utilize a standard tissue
culture incubator, with 5 % CO 2 , humidifi ed environment at
37 °C. Lentiviral vector production should be carried out in a
Biosafety Level 2 (BSL2) approved facility. We provide recipes for
all media and solutions described in the methods in Sect. 3 .

Table 3
Components and concentrations of culture media, buffers, and other solutions

Volume Components Final concentration

Complete DMEM media
440 ml

DMEM (SIGMA, # D6546)

50 ml Newborn Calf Serum (Heat Inactivated) (SIGMA #N4762-500ML) 10 %

5 ml Pen/Strep (100×, SIGMA # P4333) 1×

5 ml L -glutamine (100×, SIGMA # G7513) 1×

Induction media
480 ml DMEM (Sigma, UK # D6546)

10 ml Newborn Calf Serum (Heat Inactivated) (SIGMA #N4762-500ML) 2 %

5 ml Pen/Strep (100×, SIGMA # P4333) 1×

5 ml L -glutamine (100×, SIGMA # G7513) 1×

Induction solution (always prepared fresh)
5 ml

Sodium Butyrate 1 M solution 10 mM

495 ml Induction Media
Polybrene 100×
40 mg Hexadimethrine bromide (Polybrene) (Sigma, #107689) 40 mg

50 ml DULBECCO'S PHOSPHATE BUFFERED SALINE (PBS)

(SIGMA, # D8537)

50 ml

Sodium butyrate (NaBut 1 M)
0.55 g Sodium Butyrate (ALDRICH, # 303410) 1 M

5 ml Cell culture grade H 2 O (SIGMA, # W3500)
TSSM formulation buffer
0.12 g Tromethamine (SIGMA, # T6687) 20 mM

0.29 g NaCl (SIGMA, # S7653) 100 mM

0.5 g Sucrose (SIGMA, # S9378) 10 mg/ml

5 ml Mannitol (SIGMA, # M9546) 10 mg/ml

Up to 50 ml Cell culture grade H 2 O (SIGMA # W3500)
TVM1 media (pH 7.10)
100 ml Cell culture grade H 2 O (SIGMA # W3500)

1.38 g DMEM-H powder (SIGMA D5648-SL)

2.5 ml 1 M HEPES (SIGMA H3537)

(continued)