I had the privilege to collaborate with Dr. Heinz Arnheiter, who discovered the gene of MITF, for a few years. When I joined NCI for postdoc research in 2005, Heinz, at National Institute of Neurological Disorders and Stroke (NINDS) at that time, was already a renowned name in pigment cell and melanoma research.  Not surprisingly, he was also well-recognized as an expert in eye development, since Heinz has done a lot works on how MITF regulates pigment cells and retina formation in eyes.   

During those years a group of researchers in NIH organized a "Pigment Cell Interest Group", these included Heinz, Vince Hearing (NCI), Bill Pavan (NHGRI), Tom Hornyak (NCI), Silvio Gutkind (NIDCR), my PI Glenn Merlino (NCI), etc. Every some time there was a naughty seminar organizer who would send invites for "PIG" meeting, so eventually the group was renamed as "Pigment Cell and Melanoma Interest Group". In quite a few seminars Heinz talked about the complicated regulation of MITF expression. At transcriptional level, it has so many different alternative spliced forms. At functional level, its function requires a Goldilocks range of expression, not too high, not too low.

MITF is downregulated in melanoma but still required for the survival of melanoma cells. Glenn wanted to build a mouse model to study dose effect of MITF gene on melanoma phenotype, so this job fell onto me.  His idea was "simple": Vga-9 mice lacked pigment cells because they are deficient of Mitf expression. Therefore, if we inserted an inducible Mitf transgene into the genome, we could rescue melanocyte development by turning on the transgene in melanocyte precursor cells of Vga-9 mice.  By turning it on more or less, we can study the dose effect of Mitf gene. This indeed was conceptually straightforward, so I took the job. Little did I know that this would become the longest project in my career. 

The very first issue came even before we started anything. We chose to target the transgene to Dct+ cells, as Dct was the best known melanocytic lineage marker. However, Heinz had shown that Dct was also the downstream gene to Mitf in melanocytes, so we created a chicken-or-egg dilemma. Nevertheless, Glenn wanted to gamble on the lineage marker side of Dct, so we imported Vga-9 C57BL/6 mice from David Fisher ("Mr. MITF") at Harvard.

To make this long story short, it took me one a few months to make the inducible Mitf vector, a few months to select mouse ES cells transfected with the vector, and then a few months to generate inducible Mitf C57BL/6 mice. Not bad. All we had to do now was to breed this transgenic mice to Vga-9 mice and Dct-rtTA mice to get inducible Mitf, Dct-rtTA Vga-9 ("triple transgenic") mice. After two to three years, we were totally beaten by the low fertility of Vga-9 mice and  produced zero triple transgenic mouse. Those were good years in NCI, and I had some other projects going well, so Glenn and I decided not to give it up. He suggested to make transgenic mice by microinjection of the transgen vector into Vga-9 mouse embryos directly. We gave a try and it worked, so we reduced the breeding scheme from among-three-mice to between-two-mice. The fertility of transgenic Vag-9 mice was still pathetic; in the process we had to outcross them with C3H mice to recover some of the breeding efficiency. After eight years, finally we got the triple transgenic mice.

I asked the technician to put those mice on doxycycline (dox) diet to turn on the Mitf in the embryo. When the pups born for the first time weaned, I was so excited to run to mouse facility to check them. They were all albino. I thought many possibilities what could go wrong, trying to do trouble shooting for many ways, including full, detailed necropsy to find any little trace of rescued melanocytes (e.g. in ear/pawns/tail). Not even a single dark spot anywhere. This continued for a year during which more litters of pups were born, so I was ready to give them up. Before I said good-bye to them, I went to the mouse facility to give them the last check.   

Vga-9 mice had small, mostly closed, red eyes. However, when I starred at the triple transgenic mice full-time on dox diet, some of them starred me back with wide-open, darker eyes. I thought it might be my wishful thoughts, so I asked the technician to mark the mice with open, darker eyes. They had the same selection like mine. It was not only in my head! We rescued pigment cells in the eyes of Vga-9 mice! Later histological analysis confirmed it. Therefore, we learned that Dct persisted in the lineage of pigment cells in the eyes, but not in the lineage of cutaneous melanocytes.

As the next step, we have to test if the function of the eyes was also restored, in addition to their morphology. How would we know if a mouse can see or not? There are two approaches. The first one is measuring mouse response to moving images. We could place a mouse in font of a screen that displayed a film of moving object, and video-recorded the moves of head and eyeballs of the mice. If the mouse movement was in accord with the moving object on the screen, that would prove that it could see. We never studied mouse behaviors in our research so had no such equipment. I came up with the idea of a replacement: I could buy a mini disco ball light from Amazon, place it in front of our mice, and use cell phone to record head movement when turning on the disco ball in the dark.

When I shared the idea with our technicians, they laughed so hard: "Sure, I will display some '80s dance music at that moment. Let's dance and party with the mice!" 

My idea could not reach technical precision necessary for the measurement, so we killed it very quickly. We turned to the second approach: electroretinograms with or without flash light, by collaborating with a true expert in this field: Kapil Bharti at National Eye Institute. In fact, Kapil was the former staff scientist in Heinz's lab.

After ten years since starting this project, the results were finally published (https://iovs.arvojournals.org/article.aspx?articleid=2719516#207285500). It is not a splendid study published at a top-tier journal and cited by hundreds of people. However, it has a unique position in my career. It taught me about the complexity of life, and how this distinguishes biology from other branches of science.