Genotyping For Trps1 Knockout Mice

Credit: Dr. Shivdasani, http://hms.harvard.edu
I. Conditions for genotyping by genomic touch-down PCR:
94° – 30 sec
65° – 30 sec 5 cycles
72° – 30 sec
94° – 30 sec
60° – 30 sec 5 cycles
72° – 30 sec
94° – 30 sec
58° – 30 sec 5 cycles
72° – 30 sec
94° – 30 sec
56° – 30 sec 10 cycles
72° – 30 sec
94° – 30 sec
54° – 30 sec 25 cycles
72° – 30 sec
** Add 0.5 ul of DMSO per 25 ul reaction. Apart from that the PCR is set up as usual.
** Primers to be used for PCR:
Wild-type allele: 5’KO(1F) and 5’KO E14 (R); expect a band of about 600bp
5’-AGTGTTCCAGTCTCGAAGCCA-3’ and 5’-GGTCTGAATTGCGCAAACACC-3’
Mutant allele: 5’KO(1F) / 022 Neo EP2; expect a band of about 900 bp
Same forward primer as above, and 5’-ATCAGGATGATCTGGACGAAGAG-3’
II. probes for genotyping by Southern analysis:
For 5' probe use plasmid P80 and amplify using primers: KO(P)5'1F and KO(P)5'1R.
5’-GGCCATGGGAAGTTGTGTATT-3’ and 5’-CAAGTGGCTTAACATTCAGAG-3’
(Use P81 for 5' southerns; this is the digested probe fragment – use 1 μl).
For 3' probe use plasmid P80 and amplify using primers: KO(P)3'1F and KO(P)3'1R.
5’-GAAGTTGGGAGAACAAATAGGA-3’ and 5’-TCCTCTCAGATTGCCCTCTTTA-3’
(Use P82 for 3' southerns; this is the digested probe fragment – use 1 μl).
Enzyme WT TRPS1 Mutant TRPS1 Probe
BamHI 7 kb 5.5 kb 5’
BamHI 9 kb 8.6 kb 3’
ECORV 10 kb 5.8 kb 5’
KPNI 12 kb 11.4 kb 3’

Megakaryocyte Culture From Mouse Fetal Livers

Credit: Dr. Shivdasani, http://hms.harvard.edu
The protocol for culturing mouse megakaryocytes is essentially as described in the
following publication:
Lecine et al., Characterization of the transcription factor NF-E2 …..
J. Biol. Chem. 273:7572-7578 (Issue of March 27, 1998)
Briefly, whole livers are recovered aseptically from mouse fetuses between
embryonic days 13 and 15. We’ve typically had best results with E13.5 cultures,
which yield a purer population of MKs; although the overall cell yield is higher
with E14.5 cultures, the total number of recoverable MKs is not much higher. Fetal
liver is a significantly better source than adult bone marrow or spleen. Typically,
outbred strains of mice produce larger litters with larger livers than do inbred
mouse strains; for most purposes, either source is OK.
Single-cell suspensions are prepared by successive passage of the whole liver
through 18-, 20-, and 22- or 23-gauge needles.
Fetal liver cells are then cultured in Dulbecco’s Modified Eagle Medium
supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, 50
mg/ml streptomycin (we no longer add DMEM non-essential amino-acids), and 1%
tissue culture supernatant from a murine c-Mpl ligand producer cell line. The latter
is now our preferred source of thrombopoietin, but almost any alternate source is
probably OK at a concentration between 0.02 and 0.1 microgram/mL.
By the third day, large polyploid megakaryocytes will begin to dominate the culture,
and by the fifth day over 50% of the cell mass will be mature megakaryocytes, many
of which will be producing proplatelets, as described in the following paper:
Lecine et al., Mice lacking transcription factor NF-E2 …. Blood 1998, 92:1609-1616
(September 15, 1998 issue)
We no longer separate MKs immunologically as described in the above JBC paper;
rather, we use a one-step albumin gradient, exactly as described in Drachman et al.,
Blood 89: 483-492 (1997). We use 1.5% albumin over 3% and sediment at 1Xg for 35-
40 minutes at room temp.
Both the megakaryocyte and non-MK fractions thus obtained are useful for further
culture or for isolation of total or nuclear protein and total or poly(A)+ RNA.

Culture And Transfection Of Adherent Cells Fibroblasts Epithelial Cells

Credit: Dr. Shivdasani, http://hms.harvard.edu
DMEM (5 °C/cold room)
Dulbecco's Modidied Eagle Medium
(500mL) 11995-065 Gibco
L-glutamine (-20 °C) L-glutamine (200mM) 25030-081 Gibco
Pen+step (-20 °C) Penicillin-Streptomycin 15140-122 Gibco
FBS (-20 °C) Fetal Bovine Serum (500mL) sh-30071.03 Hyclone
Cell/Tissue culture grade dish large Tissue Culture dish 353025 Falcon
Cell/Tissue culture grade dish small Nunclon TC dish 150079 Nunclon Delta
Sterile disposable pippettes 2mL serological pippette 2mL 357507 Falcon
Sterile disposable pippettes 5mL serological pippette 5mL Falcon
Sterile disposable pippettes 10mL serological pippette 10mL Falcon
Sterile disposable pippettes 25mL serological pippette 25mL Falcon
Falcon 15mL tubes 15mL Polypropylene Conical tubes 352096 Falcon
Hanks Buffer Hanks Balanced Salt Soln. 14170-112 Gibco
Trypsin Trypsin-EDTA 25300-054 Gibco
A. Thawing and initiating cell cultures
1. Prepare cell culture medium (DMEM+) (DMEM w/ 10% FBS, 50 U Pen-Strep.,
and 2 mM L-glutamine): Add 5 mL Pen-Strep stock, 5 mL L-glutamine stock, and
50 mL FBS to 450mL of DMEM.
2. Pre-warm DMEM+ in 37 deg C water bath.
3. Prepare tissue culture-grade dishes for cells by adding DMEM+, 25mL/large dish
or 10mL/small dish.(Perform under hood using sterile technique).
4. Prepare 15-mL Falcon tube for wash by adding 10 mL of pre-warmed DMEM+.
5. Obtain cells from liquid nitrogen tanks. (Location of cells described in liq. N2
binder) and thaw vials quickly in 37°C water bath until tiny piece of ice remains.
6. Transfer cells into a 15mL Falcon tube containing DMEM+ and wash by
centrifugation for 5min at 1000 rpm. Decant supernatant by vacuum aspiration
and add fresh DMEM+ to a final volume of 2mL/large dish (i.e. if you plan on
making 3 large dishes fill to a total vol. of 6mL).
7. Add 2mL of cell suspension to each dish with DMEM+. Swirl dishes slowly in a
figure-8 path for 1 min.
8. Incubate in TC incubators (37 deg C, 5% CO2).
Notes:
1. Use sterile technique: All handling in laminar flow hood with sterile disposable
pipettes.
2. In our lab’s current stocks, 1 tube of 1mL of cryopreserved cells may be cultured
in 3 large dishes or T75 flasks (2mL/dish of washed cells). The size of the dish
and the amount of cells aliquoted depends on how many cells you want and the
amount of time the cells may grow before achieving the desired density.
3. Bring all soln. At least to R.T. (if not 37 deg C) before exposing to cells.
4. Avoid cells overshooting confluence or acidifaction of medium (clor changes to
yellow).
B. Splitting Cells (Ideal when cells cover 60-70% of dish surface area, although this
varies widely by cell type.) (Cell quality is usually maintained for 10-12 splits, or
about 1.5 months in continuous culture. Beyond this interval, it’s best to thaw
fresh stocks.)
1. Aliqoute 20mL of DMEM+ into # of large dishes corresponding to how many
times you want to divide cells. (e.g., Split 1 large dish of 293 cells @ 60%
density into 5 large dishes if you wish to harvest again in ~2days).
2. Wash Cells in Hanks’ Buffered Salt Solution (1XHBSS) or 1xPBS to remove
serum, which acts as a protease inhibitor.
3. Treat w/ Trypsin, which detaches cells from the dish and from each other.
– Remove wash Buffer by vacuum aspiration.
– Add Trypsin-EDTA to cells (3mL/Large dish, 1.5mL/small dish)
– Swirl and tap dish to detach cells (usually 2-10 min at 37°C or longer at
RT). Avoid prolonged trypsin exposure, which can be severely damaging
to cells, by periodic visual monitoring.
– After cells are fully detached, add vol.of DMEM+ so the total volume is
divisible by the number of total new plates desires 2mL/plate
(ie Add 7mL DMEM to large plate w/ 3ml of Trypsin so the tot. vol. is
10ml that will be divided into 5 new large plates (2ml/plate).
4. Mix plate by swirling and distribute cells into new plates w/ DMEM (2mL/plate)
5. Label dishes with type of cell, date split, and # of times split (Passage #).
6. Resume culture in 37°C tissue-culture incubator.
C. Lipofectin Transfection (Cell density on plates should be about 60%)
1. Prepare plasmid DNA in sterile 1mL Eppendorf tubes, Vtot/tube ~600 μL
(1 tube will be used for 1 large plate; 20ug of DNA is usually sufficient to
transfect cells in 1 large dish or T-75 flask)
DNA stocks: Ci Cf Vf (uL)
DNA eg 2μg/μL 20 μg 10
DMEM-/- 590
2. Prepare Lipofectin stock soln. in Falcon tube
3 μL of lipofectin is required for 1 μg of DNA.

Testing Rabbit Bleeds By Elisa

Credit: Dr. Mitchison, http://mitchison.med.harvard.edu
Claire Walczak
January 1996
You can test whether or not you have gotten an immune response to the peptide and how strong that immune response is by doing ELISAs against peptide conjugated to BSA. By conjugating to BSA, you will eliminate any signal for antibodies generated to KLH during immunization. Protocol:
A. Coupling Peptide to BSA
You need:
1 ml of 2 mg/ml BSA in 0.1 M NaHCO3.
1 ml of 0.2% glutaraldehyde in 0.1 M NaHCO3.
0.5 ml peptide (1 mg/ml in DMSO). Peptide can be added as a solid if soluble.
Mix, adding glutaraldehyde last. Peptide and BSA turn a little yellow even before adding glutaraldehyde.
Incubate 90' at 37 deg C.
Add 0.1 volumes of 0.1 M NaBH4 in 0.1 M NaHCO3. There will be some bubbling. Add same amount of NaBH4 after 15'. Do a quick microfuge spin if there are too many bubbles.
B. ELISA with Peptide conjugated to BSA
Coat 10 ug/ml antigen (peptide conjugated to BSA) diluted in TBS (50 ul/well) ON at 4 deg C.
Remove antigen and rinse wells 2Xs with TBST.
Block 2 hr with 200 ul of 5% NFDM in TBST.
Remove blocking reagent and rinse wells 2Xs with TBST.
Incubate in primary antibody diluted in Blocking Buffer for 2 hr at RT. I do tripling dilutions beginning at 1/10 (50 ul/well).
Remove primary antibody and rinse wells 4Xs with TBST.
Incubate in secondary antibody (1/5000 Goat anti-rabbit conjugated to AP) diluted in Blocking Buffer for 1 hr at RT (50 ul/well).
Remove secondary antibody and rinse wells 4Xs with TBST.
Rinse wells 2Xs with 50 mM HCO3; 0.5 mM MgCl2, pH 10.
Develop in 1 mg/ml p-Nitrophenyl phosphate in 50 mM HCO3; 0.5 mM MgCl2, pH 10 (50 ul/well).
Read A410 in ELISA reader.

Gene Transduction Of 1 Mks By Retroviral Infection

Credit: Dr. Shivdasani, http://hms.harvard.edu
A. BSA (Bovine Serum Albumin) step gradient:
Purify the megakaryocyte (MK) fraction from a day 2 liver culture (~10 livers/10 mL/10 cm
tissue culture dish).
1) Pipet 1.5 mL of sterile 3% BSA (made in PBS) into a 15-mL Falcon tube.
2) Slowly pipet 1.5 mL of sterile 1.5% BSA onto the 3% BSA solution.
3) Centrifuge cultured liver cells at 1200 rpm (table-top centrifuge) for 3-5 minutes.
4) Remove supernatant, loosen cell pellet by tapping gently, and resuspend in 1 mL DMEM
5) Layer this cell suspension carefully over the BSA step gradient and hold at room temp.
6) MKs will sediment within 30-50 minutes.
7) At this stage the upper layers (consisting originally of the cell suspension and the 1.5%
BSA solution may either be discarded if not needed, or washed once to remove the BSA
and cultured again to harvest the “second wave” of differentiated MKs.
8) Resuspend lower 1/3 of 3%BSA and cell pellet (consisting of the denser population of
mature MKs) in 8 mL DMEM..
9) Centrifuge for 5 minutes at 1200 rpm (table-top centriguge) to remove residual BSA, and
discard supernatant.
B. Retroviral infections:
Empirically, we have found several parameters to promote high infection rates, such as high
densities of MKs, addition of polybrene, and purity of the MK fraction.
a) Prepare 2 mL of complete DMEM with 0.5% Tpo and 2 μL polybrene (2 μL of a sterile 6
mg/mL stock solution).
b) Resuspend MK pellet in 2 mL of this complete medium.
c) Pipet 0.4 mL of the cell suspension into each well of a 12-well tissue culture plate.
d) Apply 50 μL of retrovirus stock to each desired well and resuspend.
e) Place 12-well plate into 37° C TC incubator for 24 hours.
f) After 24 hours, wash virus away as follows: Pipet 450 μL of MKs/virus into labeled
sterile Eppendorf tubes and centrifuge at 4000 rpm for 1-2 minutes. Discard the
supernatant, taking care to treat this waste with bleach.
g) Resuspend pellet (MKs) in 400 μL complete DMEM supplemented with 0.5% Tpo and
transfer cells into new wells of a 12-well TC plate.
h) Place in 37° C incubator and monitor for production of proplatelets or until cell
population achieves desired maturity.

Genomic Pcr For Alleles Of Mouse P45 Nf E2

Credit: Dr. Shivdasani, http://hms.harvard.edu
Prepare genomic DNA from tail biopsies, and resuspend in 150 μL sterile ddH2O.
Heat DNA for 15-30 minutes at 65°C to dissolve.
PCR Reaction: 10X Buffer 2.5 μL
dNTP mix (5 mM each) 1.0
Taq 0.25
NC 26 1.0
NFBS07 0.5
Neo 504 0.5
template DNA 2.0
ddH2O 17.25
total volume 25.0 μL
PCR Program: 95°C x 1 min.
94°C x 30 sec, 55°C x 1 min, 72°C x 2 min for 35 cycles
72°C x 5 min
4°C
Resolve PCR products on 1% agarose gel and visualize under UV.
Primers Neo504 and NC26 give a band for the mutant allele at 1.5 kb.
Primers NFBS07 and NC26 give a band for the wildtype allele at 530 bp.
Primers: NC26 5’ AAC TTG CCG GTA GAT GAC TTT AAT 3’
NFBS07 5’ CAC CAA ATA CTC CCA GGT GAT ATG 3’
Neo504 5’ GCC CGG TTC TTT TTG TCA AGA CCG 3’
**All primers are 50 uM**

Procedure For Culturing The Thrombopoietin Tpo Producing Fibroblast Cell Line Gp 122

Credit: Dr. Shivdasani, http://hms.harvard.edu
[Note: This cell line was produced in Paris in the labs of William Vainchenker and
Francoise Wendling, and is available for general use and distribution. The appropriate
reference to cite is Villeval et al., High thrombopoietin production by hemopoietic cells
induces a fatal myeloproliferative syndrome in mice, Blood 90:4369-4383 (1997).]
After thawing, the cells are cultured in DMEM, supplemented with 10% fetal calf serum.
Selection drugs such as G418 should NOT be added to the cultures to avoid the risk of
potential carryover into subsequent experiments.
One vial may be thawed onto one or two 10-cm dishes (or flask equivalent) and may be
split at a ratio between 1:5 and 1:10 at confluence, if desired. To optimize the concentration
of secreted Tpo, the medium should not be changed (i.e., the cells should not be re-fed)
after splitting.
Culture the cells until about 24 hours AFTER confluence is reached, and harvest the
supernatant ONCE. Additional supernatant harvests are not useful.
Filter the supernatant through a 22 micron filter and either establish the titre and use
immediately, or store at -80oC.
The working strength of each batch of supernatant should be titrated against either (a)
previous batches of supernatant, (b) a source of purified recombinant Tpo. or (c) Mpltransfected
Tpo-dependent Ba/F3 or equivalent cells.
[Note: A typical working concentration is 0.5-1%, which corresponds to about 0.1 mg/ml
purified recombinant c-Mpl ligand (Tpo).]

Staining Of Cartilage And Bone In Adult Fetal Or Neonatal Mice

Credit: Dr. Shivdasani, http://hms.harvard.edu
The key reference for this procedure is M. Jean McLeod, Teratology 22: 299-301 (1980).
Adult or neonatal mice are killed using dry ice. They are then skinned, eviscerated extensively,
and considerable effort needs to be paid to remove ALL soft tissues. Leaving soft tissue
remnants greatly compromises the quality of the data at the end of a month-long procedure!!
1) Fix mice in 95% EtOH for >5 days
2) Place in standard lab Acetone (neat) for 2 days to remove any residual fat
All the following steps are done in the dark:
3) Stain 3 days in freshly prepared and filtered staining solution (see below) at 37 degC
1 vol 0.1% Alizarian red S (in chemical drawer), in 70% EtOH (filtered)
1 vol 0.3% Alcian blue 8GS (in chemical drawer), in 70% EtOH (filtered)
1vol acetic acid
17 vol 70% EtOH
4) Wash in doubly distilled water
5) Clearing: Clear in 1% aqueous K-OH for 12-48 hrs
6) Clear for 1 week each (sequentially) in:
(20% glycerol) 1 vol glycerol/4 vol 1% KOH
(50% glycerol) 1 vol glycerol/1 vol 1% KOH
(80% glycerol) 4 vol glycerol/1 vol 1% KOH
Standard glycerol from general stores was used.
7) Store skeletal preparations in 100% glycerol in the dark
8) Photograph as needed

Mouse Islet Culture And Si Rna Protocol

(Day 1) Typically we obtain 1200 mouse islets from 12 Cd1 mice. The isolation is done by the UVA Islet Isolation Core. Islets are delivered to us in RPMI + P/S + 10% FBS with 25 mm Glucose (which is prepared by us and given to the islet core lab).
Media Recipe : RPMI (Gibco #11875-093), which contains 11.1 mM glucose. We add 5 ml Penn Strep, 50 ml FBS and 6.9 ml of sterile 1M glucose to make the final glucose concentration 25 mm
Islets are plated in 10 cm Petri dishes in RPMI media (above) and allowed to recover from isolation.
(Day 2): Islets are pooled using siliconized pasteur pipets in a sterile 15 ml blue cap tube, centrifuged 500 RPM for 1 min. Media is aspirated and pellets are gently washed in 5 ml PBS, centrifuged 1 min at 500 RPM.
Islet pellets are resuspended in serum free RPMI plus P/S such that 800 mL contains approx 150 islets and islets are plated in 6 well tissue culture dishes and incubated for 2 hrs.
Remember to plate 150 islets each for RNA and protein for the time=0. Harvest at the time of transfection.
Transfection: Desired amount of adenovirus is added to each well and incubated for 2 Hrs and then 1.2 mL serum containing media is added to each well and islets are incubated O/N. The controls with no si-RNA should also be treated the same way. Plate 150 islets in 800 m l SFM and add 1.2 ml medium later just like the transfected islets. Do not bring the control islets to the Ad-viral incubator-keep separately.
(Day 3): After 24 hrs (from the time of infection), add 1 ml of serum containing media to the islets to make the total volume 3 ml. Incubate for an additional 2 days. (Total time incubated with virus is 72 hrs). (In this protocol, we don’t remove the virus from the culture medium -we just dilute it).
(Day 5): Harvest islets in 350 ml RLT buffer/well for RNA and protein.
After 72hrs, collect the medium from the wells into a 15-ml tube. Wash the wells with 1 ml sterile PBS twice and pool to the medium. (15-ml tube now contains 3ml medium+2ml PBS). Add 350 m l RLT buffer (RNA) OR 100 m l of 1X SDS sample buffer with out blue dye (protein) to the wells and keep. (50 m l of 2XSDS buffer + 50 m l of PBS to make 1X SDS buffer, avoid blue to help measurement of protein).
Now, spin the 15-ml tube for 5 min at 1000 rpm. Remove the supernatant and resuspend the pellet in 1 ml PBS and transfer to a 1.5 ml eppendorf tube. Spin again for 5 min at 1000 rpm. Remove the supernatant and keep the pellet on ice.
Scrape the wells (RLT or SDS buffer) with a scraper and transfer the lysates to the eppendorf tubes (which contain the pellets) using a syringe. Now mix the pellet with the lysates you collect from the wells. Pass 6-7 times through the needle. Freeze the sample at –80 for later use. Confirm the knock down of Pdx1 by Western Blot and real-time RT-PCR