In vivo viral gene delivery

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14 déc. 2013 (il y a 7 années et 7 mois)

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SOP In vivo viral gene delivery Starr Lab

Title or Type of Procedure:
In vivo viral gene delivery

P. I.
Timothy K. Starr

Lab Location:

175 MoosT

Original Issue Date:


Revision Date:

Prepared By:

Timothy K. Starr

Approval Signature:

(if required by lab supervisor)

Procedural Methods and Materials:

General Safety Procedures:

handling and injection of virus
is to be performed in a Biological Safety Cabinet (BSC) using
BSL2 practices.

Two BSC hoods will be used, one in MoosT 12
175, and one in MCB 1

Wear Personal Protective Equipment (PPE): sterile gloves, eye goggles, lab coat and arm sleeves.

Gloves must be worn at all times when working with virus. Remove gloves using the ins
technique. Dispose of gloves into biohazard waste container to be autoclaved. Wash hands
immediately after removing gloves and before leaving work area. Never wear gloves outside
of the laboratory, or touch doorknobs, telephones, personal belonging
s, etc. with gloved

Transport virus by foot in a sealed, leak
proof primary container within a sealed, leak
secondary container with sufficient absorbent material between the two containers (e.g.
Screw cap tube inside a 15mL conical tube). At
tach a biohazard label to the outside listing:
name, contact address, phone #, name of reagent, emergency contact.

The most effective germicide against virus includes 30
minute exposure with 10% bleach (1:9 v/v)

Before working in BSC:

If working

in MCB 1
134E (animal facilities) post a Biohazard sign on door indicating that
unauthorized personnel may not enter during the experiment.

Remove all unnecessary equip
ment and supplies from the BSC in order to ensure smooth
s air flow.
Check that air gril
les are clear.

Turn on blower before using to remove particulates in the cabinet. Wait at least five minutes.

Wipe down surface of cabinet interior with disinfectant (paper towels soaked with 10% bleach
followed by spraying with 70% ethanol).

Place supplies and needed equipment in the BSC before beginning work to minimize the number of
movement disruptions across the air barrier of the cabinet. Only items required for the
immediate work should be placed in the BSC.

Place absorbent towels
and decontaminating solution (70% ethanol) near the BSC to facilitate quick
clean up of spills.

Wipe the exterior of supplies with a disinfectant (70% ethanol), particularly containers removed
from a water bath. Spray items with a fine mist of 70% ethano
l, this will evaporate quickly
and leave no wet mess but still be effective. Segregate items that will remain clean from the
ones that may become contaminated.

Wash hands and arms, wear appropriate protective equipment for the work being done and to
nt skin flora from contaminating your work.

Adjust stool height so that your neck and face are above the sash opening.

While working in BSC:

Delay manipulation of materials for approximately one minute after placing the hands/arms inside
the BSC. Do not r
est arms on the front grille. Raising arms slightly will lessen disruption of
air flow.

Work as far back in the cabinet as practical

at least four inches inside the front grill edge. Move
arms slowly and limit arm movement in and out of cabinet.

As a ge
neral rule of thumb, keep clean materials at least one foot away from aerosol
activities to minimize the potential for cross
contamination. The work flow should be from
"clean (left) to contaminated or dirty (right) ". Limit the movement of "dir
ty" items over
"clean" ones.

Remove media with vacuum and replace with serological pipettes.

After working in BSC:

Wipe down the surfaces of all containers and equipment with an appropriate disinfectant (70%
ethanol) and remove from the BSC.

Wipe down the

cabinet interior with disinfectant (paper towels soaked with 10% bleach followed by
spraying with 70% ethanol.

Leave blower on for several minutes with no activity so that any airborne contaminants will be
purged from the work area.

Remove gloves and disp
ose in Biological Hazards bag and wash hands.

Tips to prevent contamination:

Clean water baths frequently and/or treat water in bath.

Clean the inside of incubators frequently, particularly the water tray. Use treated di water in the
bottom of the incubat
or or water tray.

Use HEPA filters on incubator CO2 and air intake lines. Replace regularly.

Lab coat sleeves can introduce contaminants to biological safety cabinets and incubators. Use coats
designated for working in the biological safety cabinet or tiss
ue culture area, launder
frequently. Use disposable sleeve guards if contamination has been a problem.

Never pour media, remove with vacuum and replace with disposable pipettes.

Do not leave flasks of waste media in cabinet, clean after every use.

On a reg
ular basis, decontaminate under the air grilles and wherever parts are removable. Media is
commonly splattered on the front grille allowing fungus to grow undetected on the under
surface of the grille.

Decontaminate the surface of carts or trays used to t
ransfer culture flasks between the incubator and
the biological safety cabinet or microscope.

Keep pipette aids cleaned, especially the nosepiece, and replace filters regularly.

Clean and disinfect vacuum tubing. This should be done by sucking up the disin
fectant we place in
the bottom of the waste flask after we have emptied it and reattached it.

Keep the water in the incubator's water jacket full. If water levels in the jacket drop, the ceiling of
the incubator will be cooler causing condensate to form. W
ater then drops onto shelves and
cell culture containers.

Check port plugs and septums for contamination in incubator interior; they may trap moisture and
harbor fungi.


Mice in SPF microisolator cages

Sterile stock solution of


3 ml ketamine hydrochloride (100 mg/ml)

(Final conc = 11.5 mg/ml)

1.6 ml xylazine hydrochloride (100 mg/ml)

(Final conc = 6.1 mg/ml)

1.5 ml acepromazine (10 mg/ml)

(Final conc = 0.57 mg/ml)

20 ml sodium chloride (0.9%)

Sterile stock solution
of 2º anesthetizing reagent

3 ml ketamine hydrochloride (100 mg/ml)

1.5 ml acepromazine (10 mg/ml)

18.4 ml sodium chloride (0.9%)

Recombinase Adenovirus. Titer of virus is generally between 1 x 10^7 to 1 x 10^10

(1 x 10^4 to 1 x
10^7 PFU/µl)
. Virus is purchased from a commercial
source such as Eton Biosciences (Cat #
) or Cell BioLabs (Cat #
005). The virus is NOT replication competent and contains the Cre
Recombinase gene.

Bottle of 70% ethanol

23g needles and 1 m
l syringes for injection of anesthesia

30g needles and 1 ml syringes for injection of virus

Sterile pads and gauze


Follow procedure for using BSC listed above.

Place all materials in BSC, including

Weigh mouse

Load 23G syringe with
appropriate volume of 1º anesthetizing reagent (9 µl per gram
body weight = 225 µl for a 25 g mouse

= 1.3 mg/kg ketamine = 0.68 mg/kg xylazine
hydrochloride = 0.06 mg/kg acepromazine

Load 30G syringe with 10 µl of virus (between 1 to 10 x 10^5 PFUs).

esthetics procedure

Firmly hold mouse by scruff of neck pinched between forefinger and thumb, with tail
tucked under little finger.

Insert needle about 10 mm ventrally from rear leg, taking care to avoid nipples and inject

anesthetizing reagent

into the

intraperitoneal cavity

Wait until mouse is sufficiently sedated. Mouse should not have any reflex when toe is
pinched. If surgery lasts longer than 20 minutes, make a second injection
using the 2º
anesthetizing reagent (ketamine and acepromazine only).


Lay animal dorsal side up on sterile gauze with head facing away, tail towards you

Shave or wet fur with 70% alcohol at incision point.

Lift wetted skin using forceps and make small incision in the skin (not the peritoneal
wall) with scissors at
dorsomedial position and directly above the ovarian fat pad.
The fat pad should be visible beneath surface of peritoneal wall. Fat pad is
recognizable by its white color in contrast to the dark pink tissue surrounding it.
Make a second incision in the p
eritoneal wall just above the fat pad.

Place a sterile soaked saline gauze pad on the midline adjacent to the incision. Locate
ovarian fat pad and pull it out and rest it on the gauze. Stabilize the ovary by
clamping fat pad with a bulldog clip.

Under a
dissecting microscope, position ovary as to allow for insertion of a 30 G needle
into the oviduct tubule bend leading to the bursa. The needle should be inserted close
enough to the infiundibulum to see it under the bursa and the ovary. When needle is
serted into proper position, it should be visible under the bursa.

Gently push plunger of syringe to inject between the bursa and the ovary while the
syringe is position at the injection site. This works best with two people. One person
holds the needle
in place while the second person pushes the plunger. A maximum of

20 µl can be injected.

Remove the needle quickly to seal puncture site, but gently enough not to tear the bursa
and tubule. Bursa should appear to be slightly distended with proper in

Release fat pad from bulldog clip and gently replace ovaries into peritoneal cavity using
forceps. Gently close body wall by pulling the upper peritoneal lining over the lower
lining. Optional to close incision in peritoneal wall with 2

4 sutu
res using 6
0 silk
or Vicryl sutures.

Close skin with surgical staples or wound clips.

Place animal back in cage with a heat source to avoid hypothermia and speed up

Post Surgery

Mice will be m

for wellness, palpable tumors and any

complications such as organ failure, thrombosis or ischemia. Incision will be
monitored for swelling, exudate, pain or dehiscence.

Antibiotics will be administered if there are post
procedural infections, according to RAR

Ibuprofen will be administered if there are signs of pain. Administration will be 10 ml
Children’s Motrin in 500 ml drinking water and maintained daily until signs of pain
are gone.

Remove staples or wound clips 7 or more days post surgery.

Sacrifice mice

when moribund, at tumor endpoint, or at 18 months. See original protocol
for detailed description of endpoints and signs of distress

operative Record Keeping

For all surgical and anesthetic procedures, a real time procedural record

be kept and
maintained as part of the animal’s records. Records
be maintained for three years
to cover

the life of the protocol.

Records will include:

* Animal or group identification, protocol number, and the date of the

(identical sur
geries on multiple
animals will

be listed in one


* All drugs or fluids administered, including dose, route, time, and the

identity of the
person administering the drugs,

* A description of the surgical procedure and identification of the


* Ongoing findings during monitoring (eg.
Heart rate, depth of anesthesia
). For
surgeries on more than one


conducted during the same surgical setup period,
findings may

be noted after the end of the surgery.

* Notation of any var
iations from the normal and expected events during

the anesthetic
and recovery periods, including the actions taken and the

time performed, the
animal’s response to these actions, and the identity of

the person performing these

* Assessment fo
r pain and distress,

* Actions taken to alleviate pain and distress, including

interventions, and the response to these actions,

* A notation defining the end of the monitoring period (euthanasia or

recovery from the se
dation or anesthesia), including the time,

date, and the identity of
the person making the notation.

Surgical Monitoring Records

Post operative records

include the time of monitoring, any observed

problems, what
type of analgesics/antibiotics
are given, if any (including

dosages and route), whether
the animals are active, eating, drinking and

whether the incision site is healing well or
is infected, animal posture,

fur coat texture (indicators of pain), urine and feces
production. These



be kept for at least 3 days post
surgically for minor

surgical procedures or if the surgery is more invasive, until the post

operative period
is at an end (for example, when sutures are removed and

surgical wounds are
adequately healed). All records


be readily

available to the personnel involved in
post surgical monitoring, the

veterinary staff, the IACUC, and federal regulatory

Hazard Identification and Risk of Exposure to the Hazards:

Two main hazards exist: Sharps and
exposure to adenovirus

Sharps, especially needles used for injection. Never recap needles, but place directly in
Sharps container.

Adenovirus background information

[Adapted from information provided by the University of Texas Health Science Center at
Houston, Office of Research:

General Information on Human Adenovirus

There are approximately 50 different seroty
pes of human adenoviruses. Some can induce
a spectrum of illnesses including acute, self
limiting pharyngitis (a common cold),
keratoconjunctivitis (pink eye), and diarrhea. In rare cases, human adenovirus may cause
hepatitis (inflammation of the liver), o
r inflammation of other organs. Also, in rare
situations when an individual is already seriously immunocompromised (has weakened
defenses against infections, i.e. AIDS patients), the outcome could be a blood infection.
However, in most instances, adenoviru
ses cause only a common upper respiratory illness
known as a "cold." One of the most common adenovirus serotypes that infect the human
respiratory tract is Adenovirus Type 5. This is the serotype that recombinant adenoviral
vectors are currently derived fr

The epidemiology of adenovirus indicates that it is found worldwide with high
occurrences in children under the age of 5 years. In tropical regions, adenovirus
incidences usually occur in wetter areas, while temperate regions experience seasonal
rences with the highest incidences occurring in the fall, winter, and early spring.

The mode of transmission of adenovirus is directly by oral contact or aerosolized droplet
exposure to the mucous membrane with an incubation period of 1 to 10 days in heal
humans. There are no specific antiviral drugs currently available, but adenovirus shows
susceptibility to chemical disinfectants such as 1% sodium hypochlorite and 2%

A question is occasionally raised by individuals with regard to the
potential problem of
an investigator suffering from a cold while working with adenoviral vectors. Most adult
individuals have had natural adenoviral infection early in childhood, which results in
immunity to subsequent adenoviral infections. If Biosafety L
evel 2 precautions are
strictly followed, there is a very low risk of self
contamination with the vector.

Recombinant Adenoviral Vectors

made recombinant adenoviral vectors are derived from the type 5 adenovirus.
Common deletions in adenovir
al vectors include the E1 and E3 regions. The E1 deletion
renders the virus incapable of autonomously reproducing itself and the E3 deletion makes
the virus more susceptible to the human immune defense system and also provides an
area for transgene inserti
on. The E1 deletion is replaced with an "expression cassette"
that consists of a promoter, research gene and poly A signal. The recombinant vector can
be produced to very high titers in Human Embryonic Kidney (HEK) 293 cell
s (the titer
can reach up to 10^
2 infection units per millimeter).

There are several points that must be kept in mind with regard to recombinant vectors:

* Despite the fact that humans are probably immune to this vector, a vector may still
infect an individual if he/she is exposed
to a high titer. Nonetheless, theoretically,
recombinant adenoviral vectors would not replicate.

* While exposure to wild type, replication competent adenovirus may be a low risk, the
risk of exposure to recombinant adenoviral vectors is unknown. Moreo
ver, the potential
risk of exposure to different recombinant adenoviral vectors may not be the same. It is
believed that some vectors may have minimum risk (e.g., null and LacZ vectors) while
others (e.g., interleukin, TNF, or immune effector vectors) may
pose a higher risk.

Based on the current understanding, recombinant adenoviral vectors have been classified
as Class I (minimum risk) and Class II (potentially higher risk). The safety conditions
under which these two classes are to be used are very
similar, that is, generally all
procedures are performed under Biosafety Level 2 (BSL
2), while the Class II vectors are
performed under Biosafety Level 2 with the possible addition of Biosafety Level 3
practices and/or equipment.

The classification of ad
enoviral vectors into classes is only intended as a guide. Based on
clinical data, the wildtype virus (adenovirus type 5) was categorized in the Class I
classification. All other categorizations are less clear. The attenuated viruses with the E1
region del
etions might naturally be expected to be less dangerous, due to their reduced
capacity for autonomous viral replication, and categorized as Class I. Based on transgenic
animal studies, vectors expressing transgenes such as markers (e.g., LacZ, neomycin
sphotransferase, and chloramphenicol acetyl transferase) are grouped in Class I. On
the other hand, vectors expressing a product that is known to be toxic or involved in the
regulation of cell growth should be grouped in Class II. As alluded from the above
, if the
recombinant vector being studied has minimal safety data, or there is concern over the
expression of the transgene(s) or the location of the transgene(s) expressed, a
conservative safety approach should be taken and the use of Biosafety Level

2+ practices
should be used when appropriate.

[Adapted form information provided by the University of Kentucky EH&S Biological

• Adenovirus is a pathogen of respiratory and gastrointestinal mucous and


• Adenovirus (replication deficient and replication competent) can cause corneal and
conjuctival damage. Eye protection (goggles) must be worn when working with this

• Adenovirus (unlike HIV or herpes) is quite stable. After h
aving been extracted with
ether and/or chloroform, it can still be infective.

• The replication
defective virus may be complemented in vivo thereby causing the
vector to become replication competent.

Symptoms of Exposure

Any of these symptoms may occur fo
llowing adenovirus exposure:

• Acute respiratory illness (cold like symptoms)

• Pneumonia

• Conjunctival infection (red eye)

• Corneal inflammation leading up to scarification

Exposure Controls Specific to Above Risk of Exposure:


Lab coats, face

shield, gloves, surgical mask and sharps containers.

Use Biologic Safety Cabinet during preparation and handling of

All sharps and glass waste will be disposed of in an approved hard plastic sharps
container (U Stores # CX40245, MS07407 or similar),

No Cardboard sharps pouches.

If exposure to Virus occurs:

Consult a physician. Show the MSDS to the doctor in attendance. Move out of dangerous
area. If breathed in, move person into fresh air. If not breathing, give artificial
respiration. Consult a ph
ysician. In case of skin contact wash off with soap and plenty of
water. Consult a physician. In case of eye contact flush eyes with water as a precaution.

If swallowed never give anything by mouth to an unconscious person. Rinse mouth with
water. Consul
t a physician. In case of accidental injection seek medical attention

In general, decontamination is done using bleach. Keep near the hood a bottle of freshly
10% bleach (1:9 v/v) solution
and a 70% ethanol spray bottle and a bottle of
dH2O for rinsing

Decontamination of liquid waste (conditioned medium and virus containing samples)
should be performed in final
10% bleach (1:9 v/v) solution
for 30 minutes. It may
then be sewered followed by copious amounts of water.

Wear a face protectio
n shield (prevents splashes to your face while decontaminating).

Waste Generated and Disposal Methods:

Liquid waste will be collected in a flask or beaker containing bleach 10% (v/v) and will
soak for 30 minutes before being sewered.

Solid waste decontamination

Each used pipette, plate, dish, tube and tip has to be washed with
10% bleach (1:9 v/v)

before discarding it to the biohazard bag. Alternatively you can dip the used
pipette in a box containing
10% bleach (1:9 v/v) sol
(for example sharps box)
provided that enough volume will be used to cover 20% of pipette height.

Used tips should be soaked in a plastic bottle (for example, cleaned used medium bottle)
10% bleach (1:9 v/v) solution

Used plates/flasks

have to be decontaminated with
10% bleach (1:9 v/v) solution
for 30

Discard all decontaminated solid waste in the biohazard bag for incineration.

Sharps containers will be sealed when ¾ full and placed in designated waste area.

Refer to the Bio
logical Waste Disposal procedures posted on the Tissue Culture room
door for more information

Spill and Accident Response Procedures:

For spill, splash or aerosol clean up:

In general, decontamination is done using bleach. Keep near the hood a bottle of
10% bleach (1:9 v/v) solution
and a 70% ethanol spray bottle and a bottle of
dH2O for rinsing

Decontamination of liquid waste (conditioned medium and virus containing samples)
should be performed in final
10% bleach (1:9 v/v) solution
for 30 m
inutes. It may
then be sewered followed by copious amounts of water.

Decontaminating a small volume spill, large volume spill and any splashes

Wear a face protection shield (prevents splashes to your face while decontaminating),

Cover the spill with paper

towel and gently pour on top
10% bleach (1:9 v/v) solution
30 minutes, followed by a rinse with water to remove remaining bleach that may pit
or etch work surfaces & equipment if needed, then followed by a 70% ethanol rinse

Collect the paper towels to

the biohazard bag.

Refer to the Biological Decontamination and Spill Clean
up Plan posted on the Tissue
culture room door for more information

(special record keeping

such as inventories for toxins
, reporting,
that may be


For further information view the UMN DEHS website containing Bio Basic
Fact Sheets


For general information on Biosafety,
access the Biosafety in Microbiological and
Biomedical Laboratories (BMBL) 5th Edition from the CDC at


For Material Saf
ety Data Sheets access the Public Health agency of Canada website