A research on the efficiency of T-cell marking by the use of the Lck-1 promoter.

1. Nnah Austin H.
Characterization of GFP
expression in Lck-1
transgenic mice

2. Why this research is relevant?
T-lymphocytes play a paramount role in both physiological and pathogenic processes. Their
migration in itself is significant in these processes.
Central-peripheral migration
Beginning from the development of the progenitor cells from HSCs in the bone marrow, their
migration to the thymus – expansion and differentiation of their progenitor cells to immature
thymocytes – positive selection and negative selection – mature thymocytes and finally migration to
peripheral tissues.
Peripheral-peripheral migration
• Physiological: occurs during normal response to antigenic stimuli
• Pathological: occurs when T-cells are abnormally activated and migrate in response to abnormal
antigens- IBDs, T-cell lymphomas, T-cell deficiency disorders, etc.
Agenda

3. Agenda
Why is this research relevant?
In order to study T-lymphocyte migration in vivo in several disease state, we need a marker which is
specific to T-cells.
So why not use anti-CD3? (a general T-cell marker)
WITH LCK-1 GFP TRANSGENE, THERE IS NO NEED FOR ANTIBODY LABELLING.

4. Background
Lck-1 GFP transgenic mice is a gene modified mice that has a GFP
gene linked to the Lck-1 promoter
GFP
GFP is a fluorescent protein consisting of a Beta-barrel polypeptide concealing a chromophore.

5. Background
GFP (contd)
It is a bioluminescent and fluorescent non-endogenous protein first isolated from jelly fish –
Aequorea victoria

6. Background
By linking the GFP gene to a T-cell specific promoter (Lck-1 promoter)
GFP is expressed specifically in T-lymphocytes.
Lck (Lymphocyte Kinase):
Lck is a tyrosine kinase involved in signal transduction in T-cells. It
phosphorylates the CD3 which leads to ZAP-70 activation culminating
in T-cell activation, differentiation or apoptosis.
Lck has two promoters: Lck-1 (proximal) and Lck-2 (distal)
It's gene is constitutively expressed. Therefore, an Lck-1 GFP transgene
will be expressed constitutively also. GFP can now be analyzed
specifically in T-cells.

7. Objective
To demonstrate the specificity of GFP expression in T-lymphocytes of
newly generated Lck-1 GFP transgenic mice

8. Method
Flow cytometry
1. Blood collection
2. Isolation of lymphoid organs
Spleen, axillary and mesenteric lymph nodes were isolated from Lck-1 transgenic mice and a
control mice.
3. Homogenization of tissue samples
Homogenize smearing the tissues using two glass slides. Filter the cells from the tissue debris with
cotton wool.

9. Method
4. Centrifugation and re-suspension of lymph node cell sediments in 10Ml
PBS - 1000rpm, 5mins
5. Ficoll centrifugation on the spleen and blood
1Ml Ficoll solution, 2000rpm, 20mins
6. Cell counting
10^6 cells are required
7. Antibody labeling with anti-CD3 and anti-B220
Anti-CD3 is a T cell general marker. It is conjugated with a fluorescent dye – APC CY7A (red dye)
Anti-B220 is a B cell general marker. It is conjugated with a fluorescent dye – PE CY7A (green dye)
8. Flow cytometry analysis

10. Observation
Flow cytometry (Spleen- Lck-1 GFP)

11. Observation
Flow cytometry (Spleen- Control)

12. Observation
Flow cytometry (lymph node- Lck-1 GFP)

13. Observation
Flow cytometry (Lymph node- control)

14. Observation
Flow cytometry (blood)

15. Observation

16. Method
Immunohistochemistry
1. Isolation of lymphoid organs
spleen and lymph node from an Lck-1 transgenic mice and a control mice.
2. Frozen section block preparation
3. Micro-cutting of frozen tissue block
drying for one day
4. Fixation with acetone
5. Blocking to prevent non-specific antibody binding
Tween solution or PBS azide solution.

17. Method
6. Immunohistochemical staining with anti-CD5 and anti-B220
antibodies
Anti-CD5 is a T-cell marker. It is conjugated with a fluorochrome – PE
Anti-B220 is a B-cell marker. It is conjugated with a fluorochrome – Alexa 640
6. Fluorescent microscopy

18. Results
Immunohistochemistry (spleen)

21. Observation
Immunohistochemistry (lymph node)

24. Conclusion
From our developed Lck-1 transgenic mice, we could demonstrate that
GFP was specifically expressed in T-lymphocytes.
What next?
Lck-1 GFP transgene is now being used in our lab for the study of
rheumatoid arthritis in mouse models.
Collagen induced arthritis in mice -> T-cell isolation from the arthritic mice -> injection of isolated T-
cells into healthy mice -> study of T-cell migration with Lck-1 GFP transgenic mice.